Methods and compositions for modulating gene expression

ABSTRACT

The present disclosure provides compositions with a modulating gene expression and methods for modulating transcription.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of International PatentApplication No. PCT/US17/50553, filed Sep. 7, 2016, which claimspriority to and benefit from U.S. provisional application Ser. Nos.62/384,603 (filed Sep. 7, 2016), 62/416,501 (filed Nov. 2, 2016),62/439,327 (filed Dec. 27, 2016), and 62/542,703 (filed Aug. 8, 2017),the contents of each of which are herein incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 16, 2018, isnamed 2012802-0009 ST25.txt and is 25 kilobytes in size.

BACKGROUND

Many diseases are caused by defective regulation of expression ofcertain genes.

SUMMARY

Among other things, the present disclosure provides various agents,compositions, and methods for modulating gene expression, delivery to acell (e.g., a mammalian cell such as a mammalian somatic cell; e.g.,delivery across a cell membrane), and related methods of treatment. Tothe inventor's knowledge, the present disclosure provides the firstdisclosure of site-specific agents that physically disrupt and/or modifyanchor-sequence mediated conjunctions. The present disclosure alsoprovides, among other things, site-specific agents that act to disruptand/or modify anchor sequence-mediated conjunctions by genetic and/orepigenetic methods.

In some embodiments, the present disclosure provides a site-specificdisrupting agent, comprising: a DNA-binding moiety that bindsspecifically to one or more target anchor sequences within a cell andnot to non-targeted anchor sequences within the cell with sufficientaffinity that it competes with binding of an endogenous nucleatingpolypeptide within the cell.

In some embodiments, the present disclosure provides a method ofmodulating expression of a gene within an anchor sequence-mediatedconjunction that comprises a first anchor sequence and a second anchorsequence, the method comprising a step of: contacting the first and/orsecond anchor sequence with a site-specific disrupting agent asdisclosed herein

In some embodiments, the present disclosure provides a method ofmodulating expression of a gene within 10 kb of a first anchor sequencewithin an anchor sequence-mediated conjunction comprising a first anchorsequence and a second anchor sequence, the method comprising a step of:contacting the first and/or second anchor sequence with a site-specificdisrupting agent as disclosed herein.

In some embodiments, the present disclosure provides a method ofincreasing expression of a gene within an anchor sequence-mediatedconjunction that comprises a first anchor sequence and a second anchorsequence, wherein the first and/or the second anchor sequence is locatedwithin 10 kb of an external enhancing sequence, the method comprising astep of contacting the first and/or second anchor sequence with asite-specific disrupting agent as disclosed herein.

In some embodiments, the present disclosure provides methods comprisinga step of delivering a site-specific disrupting agent as disclosedherein to a mammalian cell.

In some embodiments, the present disclosure provides fusion moleculescomprising: (i) a site-specific targeting moiety and (ii) a deaminatingagent, wherein the site-specific targeting moiety targets the fusionmolecule to a target anchor sequence but not to at least one non-targetanchor sequence.

In some embodiments, the present disclosure provides compositionscomprising: (i) a fusion polypeptide comprising an enzymaticallyinactive Cas polypeptide and a deaminating agent, or a nucleic acidencoding the fusion polypeptide; and (ii) a guide RNA, wherein the guideRNA targets the fusion polypeptide to a target anchor sequence but notto at least one non-target anchor sequence.

In some embodiments, the present disclosure provides methods ofmodulating expression of a gene within an anchor sequence-mediatedconjunction that comprises a first anchor sequence and a second anchorsequence, the method comprising a step of: contacting the first and/orsecond anchor sequence with a site-specific disrupting agent asdisclosed herein.

In some embodiments, the present disclosure provides methods ofmodulating expression of a gene within 10 kb of a first anchor sequencewithin an anchor sequence-mediated conjunction comprising a first anchorsequence and a second anchor sequence, the method comprising a step of:contacting the first and/or second anchor sequence with a site-specificdisrupting agent as disclosed herein.

In some embodiments, the present disclosure provides methods ofdecreasing expression of a gene within an anchor sequence-mediatedconjunction that comprises a first anchor sequence, a second anchorsequence, and an internal enhancing sequence, the method comprising astep of: contacting the first and/or second anchor sequence with asite-specific disrupting agent as disclosed herein.

In some embodiments, the present disclosure provides methods comprisinga step of: (a) delivering the fusion molecule or composition asdescribed herein to a mammalian cell.

In some embodiments, the present disclosure provides methods comprisinga step of: (a) substituting, adding, or deleting one or more nucleotidesof an anchor sequence within a mammalian somatic cell.

In some embodiments, the present disclosure provides methods comprisinga step of delivering a mammalian somatic cell to a subject having adisease or condition, wherein one or more nucleotides of an anchorsequence within the mammalian somatic cell has been substituted, added,or deleted.

In some embodiments, the present disclosure provides methods comprisinga step of: (a) administering somatic mammalian cells to a subject,wherein the somatic mammalian cells were obtained from the subject, anda fusion molecule or composition as disclosed herein had been deliveredex vivo to the mammalian cells.

In some embodiments, the present disclosure provides fusion moleculescomprising: (i) a site-specific targeting moiety and (ii) an epigeneticmodifying agent, wherein the site-specific targeting moiety targets thefusion molecule to a target anchor sequence but not to at least onenon-target anchor sequence.

In some embodiments, the present disclosure provides site-specific guideRNAs that comprises a targeting domain complementary to a target nucleicacid comprising an anchor sequence.

In some embodiments, the present disclosure provides compositionscomprising: (i) a fusion polypeptide comprising an enzymaticallyinactive Cas polypeptide and an epigenetic modifying agent, or a nucleicacid encoding the fusion polypeptide; and (ii) a guide RNA, wherein theguide RNA targets the fusion polypeptide to a target anchor sequence butnot to at least one non-target anchor sequence.

In some embodiments, the present disclosure provides methods ofmodulating expression of a gene within an anchor sequence-mediatedconjunction that comprises a first anchor sequence and a second anchorsequence, the method comprising a step of: contacting the first and/orsecond anchor sequence with a fusion molecule or composition asdisclosed herein.

In some embodiments, the present disclosure provides methods ofmodulating expression of a gene within 10 kb of a first anchor sequencewithin an anchor sequence-mediated conjunction comprising a first anchorsequence and a second anchor sequence, the method comprising a step of:contacting the first and/or second anchor sequence with a fusionmolecule or composition as disclosed herein.

In some embodiments, the present disclosure provides methods ofdecreasing expression of a gene within an anchor sequence-mediatedconjunction that comprises a first anchor sequence, a second anchorsequence, and an internal enhancing sequence, the method comprising astep of: contacting the first and/or second anchor sequence with afusion molecule or composition as disclosed herein.

In some embodiments, the present disclosure provides methods ofincreasing expression of a gene within an anchor sequence-mediatedconjunction that comprises a first anchor sequence and a second anchorsequence, wherein the first and/or the second anchor sequence is locatedwithin 10 kb of an external enhancing sequence, the method comprising astep of: contacting the first and/or second anchor sequence with afusion molecule or composition as disclosed herein.

In some embodiments, the present disclosure provides methods comprisinga step of: (a) delivering a fusion molecule or composition as disclosedherein to a mammalian cell.

In some embodiments, the present disclosure provides an engineeredsite-specific nucleating agent, comprising: an engineered DNA-bindingmoiety that binds specifically to one or more target sequences within acell and not to non-targeted sequences within the cell with sufficientaffinity that it competes binding of an endogenous nucleatingpolypeptide within the cell; and a nucleating polypeptide dimerizationdomain associated with the engineered DNA-binding moiety so that, sothat, when the engineered DNA-binding moiety is bound at the at leastone target sequences, the nucleating polypeptide dimerization domain islocalized thereto, and each at least one targeted sequence is a targetanchor sequence wherein the at least one or more target anchor sequencesis positioned relative to an anchor sequence to which a nucleatingpolypeptide binds so that, when the nucleating polypeptide dimerizationdomain is localized to the target anchor sequence, interaction betweenthe nucleating polypeptide dimerization domain and the nucleatingpolypeptide generates an anchor-sequence-mediated conjunction.

In one aspect, the disclosure includes a pharmaceutical preparationcomprising a composition that binds an anchor sequence of an anchorsequence-mediated conjunction and alters formation of the anchorsequence-mediated conjunction, wherein the composition modulatestranscription, in a human cell, of a target gene associated with theanchor sequence-mediated conjunction.

In one aspect, the disclosure includes a composition comprising atargeting moiety that binds an anchor sequence of an anchorsequence-mediated conjunction and alters formation of the anchorsequence-mediated conjunction (e.g., alters affinity of the anchorsequence to a conjunction nucleating molecule, e.g., at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or more.

In one aspect, the disclosure includes a pharmaceutical preparationcomprising a composition comprising a targeting moiety that binds ananchor sequence of an anchor sequence-mediated conjunction and altersformation of the anchor sequence-mediated conjunction, wherein thecomposition modulates transcription, e.g., in a human cell, of a targetgene in an expression unit associated with the anchor sequence-mediatedconjunction.

In various aspects of the disclosure delineated herein, one or more ofthe various embodiments described herein may be combined.

In some embodiments, the targeting moiety comprises an effector moietythat: (i) is a chemical, e.g., a chemical that modulates a cytosine (C)or an adenine (A) (e.g., Na bisulfite, ammonium bisulfite); (ii) hasenzymatic activity (methyl transferase, demethylase, nuclease (e.g.,Cas9), deaminase); or (iii) sterically hinders formation of the anchorsequence-mediated conjunction. [e.g., membrane translocatingpolypeptide+nanoparticle].

In some embodiments, the anchor sequence-mediated conjunction isassociated with one or more transcriptional control sequences. In oneembodiment, one or more transcriptional control sequences are inside theanchor sequence-mediated conjunction, e.g., a Type 1 anchorsequence-mediated conjunction. In another embodiment, one or more one ormore transcriptional control sequences are outside the anchorsequence-mediated conjunction comprises, e.g., a Type 2 anchorsequence-mediated conjunction. In another embodiment, one or more one ormore transcriptional control sequences are inside, e.g., enhancingsequences, and outside, at least partially, e.g., silencing sequences,the anchor sequence-mediated conjunction, e.g., a Type 3 anchorsequence-mediated conjunction. In another embodiment, one or more one ormore transcriptional control sequences are inside, e.g., enhancingsequences, and outside, at least partially, e.g., enhancing sequences,the anchor sequence-mediated conjunction, e.g., a Type 4 anchorsequence-mediated conjunction.

In some embodiments, the composition disrupts formation of the anchorsequence-mediated conjunction (e.g., decreases affinity of the anchorsequence to a conjunction nucleating molecule, e.g., at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or more). In some embodiments, the composition promotesformation of the anchor sequence-mediated conjunction (e.g., increasesaffinity of the anchor sequence to a conjunction nucleating molecule,e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or more). In some embodiments, thetarget gene is inside the anchor sequence-mediated conjunction. In someembodiments, the target gene is outside the anchor sequence-mediatedconjunction. In some embodiments, the target gene is inside and outsidethe anchor sequence-mediated conjunction. In some embodiments, thecomposition physically disrupts formation of the anchorsequence-mediated conjunction, e.g., composition is both targeting andeffector, e.g., membrane translocating polypeptide. In some embodiments,the composition comprises a targeting moiety (e.g., gRNA, membranetranslocating polypeptide) that binds the anchor sequence, operablylinked to an effector moiety that modulates the formation of aconjunction mediated by the anchor sequence. In some embodiments, theeffector moiety is a chemical, e.g., a chemical that modulates acytosine (C) or an adenine (A) (e.g., Na bisulfite, ammonium bisulfite).In some embodiments, the effector moiety has enzymatic activity (methyltransferase, demethylase, nuclease (e.g., Cas9), deaminase). In someembodiments, the effector moiety sterically hinders formation of theanchor sequence-mediated conjunction, e.g., membrane translocatingpolypeptide and/or nanoparticle.

In some embodiments, the composition or method described herein furthercomprises at least one polypeptide with each comprising at least onesequence of ABX^(n)C, where A is selected from a hydrophobic amino acidor an amide containing backbone, e.g., aminoethyl-glycine, with anucleic acid side chain; B and C may be the same or different, and areeach independently selected from arginine, asparagine, glutamine,lysine, and analogs thereof; X is each independently a hydrophobic aminoacid or X is each independently an amide containing backbone, e.g.,aminoethyl-glycine, with a nucleic acid side chain; and n is an integerfrom 1 to 4, wherein the polypeptide hybridizes a nucleic acid sequencewithin an anchor sequence-mediated conjunction (e.g., anchor sequence ofan anchor sequence-mediated conjunction, e.g., CTCF binding motif, BORISbinding motif, cohesin binding motif, USF1 binding motif, YY1 bindingmotif, TATA-box, ZNF143 binding motif, etc).

The composition and method as described in various embodiments of theabove aspect may be utilized in any other aspect delineated herein.

In one aspect, the disclosure includes a method of modulating expressionof a target gene in an anchor sequence-mediated conjunction comprisingtargeting a sequence outside of or that is not part of the target geneor its associated transcriptional control sequences that influencetranscription of the gene, such as targeting an anchor sequence, therebymodulating the gene's expression.

In one aspect, the disclosure includes a method of modulatingtranscription of a target gene comprising targeting a sequencenon-contiguous with the target gene or its associated transcriptionalcontrol sequences that influence transcription of the target gene, suchas targeting an anchor sequence, to alter formation of the anchorsequence-mediated conjunction.

In some embodiments, the method comprises an anchor sequence-mediatedconjunction with one or more associated genes and one or moretranscriptional control sequences within the anchor sequence-mediatedconjunction. In some embodiments, the anchor sequence-mediatedconjunction comprises one or more associated genes and one or moretranscriptional control sequences reside outside the anchorsequence-mediated conjunction. In some embodiments, the anchorsequence-mediated conjunction comprises one or more associated genes andone or more transcriptional control sequences reside inside and outside,at least partially, the anchor sequence-mediated conjunction. Forexample, one or more repressive signals may be outside the anchorsequence-mediated conjunction and one or more enhancing sequences andthe target gene are inside the anchor sequence-mediated conjunction. Inanother example, one or more enhancing sequences reside inside andoutside the anchor sequence-mediated conjunction.

In some embodiments, the target gene is non-contiguous with one or moreanchor sequences. In some embodiments where the gene is non-contiguouswith the anchor sequence, the gene may be separated from the anchorsequence by about 100 bp to about 500 Mb, about 500 bp to about 200 Mb,about 1 kb to about 100 Mb, about 25 kb to about 50 Mb, about 50 kb toabout 1 Mb, about 100 kb to about 750 kb, about 150 kb to about 500 kb,or about 175 kb to about 500 kb. In some embodiments, the gene isseparated from the anchor sequence by about 100 bp, 300 bp, 500 bp, 600bp, 700 bp, 800 bp, 900 bp, 1 kb, 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30kb, 35 kb, 40 kb, 45 kb, 50 kb, 55 kb, 60 kb, 65 kb, 70 kb, 75 kb, 80kb, 85 kb, 90 kb, 95 kb, 100 kb, 125 kb, 150 kb, 175 kb, 200 kb, 225 kb,250 kb, 275 kb, 300 kb, 350 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb,900 kb, 1 Mb, 2 Mb, 3 Mb, 4 Mb, 5 Mb, 6 Mb, 7 Mb, 8 Mb, 9 Mb, 10 Mb, 15Mb, 20 Mb, 25 Mb, 50 Mb, 75 Mb, 100 Mb, 200 Mb, 300 Mb, 400 Mb, 500 Mb,or any size therebetween.

In some embodiments, the anchor sequence-mediated conjunction comprisesthe target gene and is associated with one or more transcriptionalcontrol sequences, e.g., silencing/repressive sequences and enhancingsequences. In some embodiments, the anchor sequence-mediated conjunctioncomprises one or more, e.g., 2, 3, 4, 5, or more, genes. In someembodiments, the anchor sequence-mediated conjunction is associated withone or more, e.g., 2, 3, 4, 5, or more, transcriptional controlsequences.

In some embodiments, the target gene is non-contiguous with one or moretranscriptional control sequences. In some embodiments where the gene isnon-contiguous with the transcriptional control sequence, the gene maybe separated from the transcriptional control sequence by about 100 bpto about 500 Mb, about 500 bp to about 200 Mb, about 1 kb to about 100Mb, about 25 kb to about 50 Mb, about 50 kb to about 1 Mb, about 100 kbto about 750 kb, about 150 kb to about 500 kb, or about 175 kb to about500 kb. In some embodiments, the gene is separated from thetranscriptional control sequence by about 100 bp, 300 bp, 500 bp, 600bp, 700 bp, 800 bp, 900 bp, 1 kb, 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30kb, 35 kb, 40 kb, 45 kb, 50 kb, 55 kb, 60 kb, 65 kb, 70 kb, 75 kb, 80kb, 85 kb, 90 kb, 95 kb, 100 kb, 125 kb, 150 kb, 175 kb, 200 kb, 225 kb,250 kb, 275 kb, 300 kb, 350 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb,900 kb, 1 Mb, 2 Mb, 3 Mb, 4 Mb, 5 Mb, 6 Mb, 7 Mb, 8 Mb, 9 Mb, 10 Mb, 15Mb, 20 Mb, 25 Mb, 50 Mb, 75 Mb, 100 Mb, 200 Mb, 300 Mb, 400 Mb, 500 Mb,or any size therebetween.

In one aspect, the disclosure includes a pharmaceutical compositioncomprising (a) a targeting moiety and (b) a DNA sequence, e.g.,comprising an anchor sequence.

In one aspect, the disclosure includes a composition comprising atargeting moiety that binds an anchor sequence of an anchorsequence-mediated conjunction and alters formation of the anchorsequence-mediated conjunction (e.g., alters affinity of the anchorsequence to a conjunction nucleating molecule, e.g., at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or more.

In one aspect, the disclosure includes a protein comprising a domain,e.g., an enzyme domain, that acts on DNA (e.g., a nuclease domain, e.g.,a Cas9 domain, e.g., a dCas9 domain; a DNA methyltransferase, ademethylase, a deaminase), in combination with at least one guide RNA(gRNA) or antisense DNA oligonucleotide that targets the protein to ananchor sequence of a target anchor sequence-mediated conjunction,wherein the composition is effective to alter, in a human cell, thetarget anchor sequence-mediated conjunction.

In some embodiments, the enzyme domain is a Cas9 or a dCas9. In someembodiments, the protein comprises two enzyme domains, e.g., a dCas9 anda methylase or demethylase domain.

The composition as described in various embodiments of the above aspectmay be utilized in any other aspect delineated herein.

In one aspect, the disclosure includes a composition for introducing atargeted alteration to an anchor sequence-mediated conjunction tomodulate transcription of a nucleic acid sequence, the compositioncomprising a targeting moiety that binds the anchor sequence.

In some embodiments, the targeting moiety includes a sequence targetingpolypeptide, such as an enzyme, e.g., Cas9. In some embodiments, thetargeting moiety includes a fusion of a sequence targeting polypeptideand a conjunction nucleating molecule, e.g. a fusion of dCas9 and aconjunction nucleating molecule. In some more embodiments, the targetingmoiety further includes a guide RNA or nucleic acid encoding the guideRNA. In some additional embodiments, the targeting moiety targets one ormore nucleotides, such as through CRISPR, TALEN, dCas9, recombination,transposon, etc., of an anchor sequence within the anchorsequence-mediated conjunction for substitution, addition or deletion. Insome embodiments, the targeting moiety targets one or more DNAmethylation sites within the anchor sequence-mediated conjunction. Insome more embodiments, the targeting moiety introduces at least one ofthe following: at least one exogenous anchor sequence; an alteration inat least one conjunction nucleating molecule binding site, such as byaltering binding affinity for the conjunction nucleating molecule; achange in an orientation of at least one common nucleotide sequence,such as a CTCF binding motif, YY1 binding motif, ZNF143 binding motif,or other binding motif mentioned herein; and a substitution, addition ordeletion in at least one anchor sequence, such as a CTCF binding motif,YY1 binding motif, ZNF143 binding motif, or other binding motifmentioned herein.

In certain embodiments, the composition modifies a chromatin structure.

In some embodiments, the composition comprises a vector comprising thetargeting moiety, such as a viral vector, e.g., a lentiviral vector.

In certain embodiments, the targeted alteration alters at least one of abinding site for a conjunction nucleating molecule, such as the bindingaffinity for an anchor sequence within the anchor sequence-mediatedconjunction, an alternative splicing site, and a binding site for anon-translated RNA.

In some embodiments, the disclosure includes a pharmaceuticalcomposition comprising the composition described herein.

The composition as described in various embodiments of the above aspectmay be utilized in any other aspect delineated herein.

In one aspect, the disclosure includes a composition comprising asynthetic conjunction nucleating molecule with a selected bindingaffinity for an anchor sequence within a target anchor sequence-mediatedconjunction.

In some embodiments, the binding affinity may be at least 10%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, orhigher or lower than the affinity of an endogenous conjunctionnucleating molecule that associates with the target anchor sequence. Insome embodiments, the synthetic conjunction nucleating molecule hasbetween about 30-90%, about 30-85%, about 30-80%, about 30-70%, about50-80%, about 50-90% amino acid sequence identity to the endogenousconjunction nucleating molecule.

In some embodiments, the conjunction nucleating molecule disrupts, suchas through competitive binding, the binding of an endogenous conjunctionnucleating molecule to its binding site. In some more embodiments, theconjunction nucleating molecule is engineered to bind a target sequence.

In some embodiments, the composition further includes a carrier, such asa polymeric carrier or targeting moiety, e.g., a liposome, peptide,aptamer, or combination therein.

In certain embodiments, the disclosure includes a method of preparingthe conjunction nucleating molecule with selected binding affinity.

The composition as described in various embodiments of the above aspectmay be utilized in any other aspect delineated herein.

In one aspect, the disclosure includes a composition comprising atargeting moiety that binds a specific anchor sequence-mediatedconjunction to alter a topology of the anchor sequence-mediatedconjunction.

In some embodiments, the targeting moiety is a nucleic acid sequence, aprotein, protein fusion, or a membrane translocating polypeptide. Insome embodiments, the nucleic acid sequence is selected from the groupconsisting of a gRNA, and a sequence complementary or a sequencecomprising at least 80%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% complementary sequence to ananchor sequence. In some embodiments, the nucleic acid sequencecomprises a sequence complementary or a sequence comprising at least80%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% complementary sequence to abinding motif for aconjunction nucleating molecule or consensus sequence. In someembodiments, the protein is a conjunction nucleating molecule, e.g.,CTCF, cohesin, USF1, YY1, TAF3, ZNF143, or another polypeptide, adominant negative conjunction nucleating molecule, a protein with aDNA-binding sequence, e.g., transcription factor, a fusion of a sequencetargeting polypeptide and a conjunction nucleating molecule. In someembodiments, the membrane translocating polypeptide comprises at leastone sequence of ABX^(n)C, wherein A is selected from a hydrophobic aminoacid or an amide containing backbone, e.g., aminoethyl-glycine, with anucleic acid side chain; B and C may be the same or different, and areindependently selected from arginine, asparagine, glutamine, lysine, andanalogs thereof; X is each independently a hydrophobic amino acid or Xis each independently an amide containing backbone, e.g.,aminoethyl-glycine, with a nucleic acid side chain; and n is an integerfrom 1 to 4. In some embodiments, the protein is selected from the groupconsisting of epigenetic enzymes (DNA methylases (e.g., DNMT3a, DNMT3b,DNMTL), DNA demethylases (e.g., the TET family), histonemethyltransferases, histone deacetylase (e.g., HDAC1, HDAC2, HDAC3),sirtuin 1, 2, 3, 4, 5, 6, or 7, lysine-specific histone demethylase 1(LSD1), histone-lysine-N-methyltransferase (Setdb1), euchromatichistone-lysine N-methyltransferase 2 (G9a), histone-lysineN-methyltransferase (SUV39H1), enhancer of zeste homolog 2 (EZH2), virallysine methyltransferase (vSET), histone methyltransferase (SET2), andprotein-lysine N-methyltransferase (SMYD2)), a fusion of a sequencetargeting polypeptide and a conjunction nucleating molecule.

In some embodiments, the targeting moiety comprises a sequence targetingpolypeptide, e.g. Cas9, a fusion of a sequence targeting polypeptide,e.g. a fusion of dCas9 and a conjunction nucleating molecule, or aconjunction nucleating molecule. In some embodiments, the targetingmoiety comprises a guide RNA or nucleic acid encoding the guide RNA. Insome embodiments, the targeting moiety introduces a targeted alterationinto the anchor sequence-mediated conjunction to modulate transcription,in a human cell, of a gene in the anchor sequence-mediated conjunction.

In some embodiments, the targeting moiety binds an anchor sequence ofthe anchor sequence-mediated conjunction and the targeting moietyintroduces a targeted alteration into the anchor sequence to modulatetranscription, in a human cell, of a gene in the anchorsequence-mediated conjunction. In some embodiments, the targetedalteration comprises at least one of a substitution, addition ordeletion of one or more nucleotides, e.g., in the anchor sequence. Insome embodiments, the targeted alteration comprises at least one of asubstitution, addition or deletion of one or more nucleotides in aanchor sequence, e.g., a binding motif for a conjunction nucleatingmolecule, such as one described herein. In some embodiments, thetargeted alteration comprises an opposite orientation of at least onecommon nucleotide sequence, e.g., a binding motif for a conjunctionnucleating molecule. In some embodiments, the targeted alterationcomprises a non-naturally occurring anchor sequence to form or disruptthe anchor sequence-mediated conjunction.

The composition as described in various embodiments of the above aspectmay be utilized in any other aspect delineated herein.

In one aspect, the disclosure includes a composition comprising aprotein comprising a first polypeptide comprising a Cas or modified Casprotein domain and a second polypeptide comprising a polypeptide havingDNA methyltransferase activity [or associated with demethylation ordeaminase activity], in combination with at least one guide RNA (gRNA)or antisense DNA oligonucleotide that targets the protein to an anchorsequence of a target anchor sequence-mediated conjunction, wherein thesystem is effective to alter, in a human cell, the target anchorsequence-mediated conjunction.

In some embodiments, the composition is effective to alter, in a humancell, the target anchor sequence-mediated conjunction.

The composition as described in various embodiments of the above aspectmay be utilized in any other aspect delineated herein.

In one aspect, the disclosure includes a pharmaceutical compositioncomprising a Cas protein and at least one guide RNA (gRNA) that targetsthe Cas protein to an anchor sequence of a target anchorsequence-mediated conjunction, wherein the Cas protein is effective tocause a mutation of the target anchor sequence that decreases theformation of an anchor sequence-mediated conjunction associated with thetarget anchor sequence.

In one aspect, the disclosure includes a synthetic nucleic acidcomprising a plurality of anchor sequences, a gene sequence, and atranscriptional control sequence.

In some embodiments, the gene sequence and the transcriptional controlsequence are between the plurality of anchor sequences. In someembodiments, the nucleic acid comprises, in order, (a) an anchorsequence, a gene sequence, a transcriptional control sequence, and ananchor sequence or (b) an anchor sequence, a transcriptional controlsequence, a gene sequence, and an anchor sequence.

In some embodiments, the sequences are separated by linker sequences. Insome embodiments, the anchor sequences are between 7-100 nts, 10-100nts, 10-80 nts, 10-70 nts, 10-60 nts, 10-50 nts, or 20-80 nts. In someembodiments, the nucleic acid is between 3,000-50,000 bp, 3,000-40,000bp, 3,000-30,000 bp, 3,000-20,000 bp, 3,000-15,000 bp, 3,000-12,000 bp,3,000-10,000 bp, 3,000-8,000 bp, 5,000-30,000 bp, 5,000-20,000 bp,5,000-15,000 bp, 5,000-12,000 bp, 5,000-10,000 bp or any rangetherebetween.

In some embodiments, a vector comprises the nucleic acid describedherein.

In some embodiments, a cell comprises the nucleic acid described herein.

In some embodiments, a pharmaceutical composition comprises the nucleicacid described herein.

In some embodiments, a method of modulating expression of a gene byadministering a composition comprises the nucleic acid described herein.

The nucleic acid as described in various embodiments of the above aspectmay be utilized in any other aspect delineated herein.

In one aspect, the disclosure includes a kit comprising (a) a nucleicacid encoding a protein comprising a first polypeptide domain thatcomprises a Cas or modified Cas protein and a second polypeptide domainthat comprises a polypeptide having DNA methyltransferase activity [orassociated with demethylation or deaminase activity]; and (b) at leastone guide RNA (gRNA) for targeting the protein to an anchor sequence ofa target anchor sequence-mediated conjunction in a target cell.

In some embodiments, (a) and (b) are provided in the same vector, e.g.,a plasmid, an AAV vector, an AAV9 vector. In some embodiments, (a) and(b) are provided in separate vectors.

The kit as described in various embodiments of the above aspect may beutilized in any other aspect delineated herein.

In one aspect, the disclosure includes a method of preparing aconjunction nucleating molecule with selected binding affinity.

In one aspect, the disclosure includes a method of (altering geneexpression/altering an anchor sequence-mediated conjunction) in amammalian subject comprising administering to the subject (separately orin the same pharmaceutical composition) (i) a protein comprising a firstpolypeptide domain that comprises a Cas or modified Cas protein and asecond polypeptide domain that comprises a polypeptide having DNAmethyltransferase activity [or associated with demethylation ordeaminase activity] or (ii) a nucleic acid encoding a protein comprisinga first polypeptide domain that comprises a Cas or modified Cas proteinand a second polypeptide domain that comprises a polypeptide has a rolein DNA methyltransferase activity [or associated with demethylation ordeaminase activity], and at least one guide RNA (gRNA) that targets ananchor sequence of an anchor sequence-mediated conjunction.

In some embodiments, the anchor sequence is or comprises a CTCF bindingmotif, such as SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, theanchor sequence is or comprises a CTCF binding motif associated with atarget disease gene.

In some embodiments, the Cas protein is dCas9; dCas9 is human codonoptimized. In some embodiments, the methyltransferase is a DNMT familymethyltransferase. In some embodiments, the polypeptide is a TET familyenzyme. In some embodiments, the protein has a linker between the firstand second polypeptide.

In some embodiments, the gRNAs are selected from gRNAs for differentdiseases.

The method as described in various embodiments of the above aspect maybe utilized in any other aspect delineated herein.

In one aspect, the disclosure includes a method of modifying a chromatinstructure, such as a two-dimensional structure, comprising altering atopology of an anchor sequence-mediated conjunction to modulatetranscription of a nucleic acid sequence. The altered topology of theanchor sequence-mediated conjunction, such as a loop, modulatestranscription of the nucleic acid sequence.

In another aspect, the disclosure includes a method of modifying achromatin structure, such as a two-dimensional structure, comprisingaltering a topology of a plurality of anchor sequence-mediatedconjunctions to modulate transcription of a nucleic acid sequence. Thealtered topology of the plurality of anchor sequence-mediatedconjunctions, such as multiple loops, modulates transcription of thenucleic acid sequence.

In another aspect, the disclosure includes a method of modulatingtranscription of a nucleic acid sequence comprising altering an anchorsequence-mediated conjunction, such as a loop, that influencestranscription of a nucleic acid sequence. The anchor sequence-mediatedconjunction modulates transcription of the nucleic acid sequence.

In certain embodiments, altering the anchor sequence-mediatedconjunction modifies a chromatin structure. For example, modifying thechromatin structure by substituting, adding or deleting one or morenucleotides within an anchor sequence of the anchor sequence-mediatedconjunction modifies the chromatin structure.

In various embodiments of the above aspects or any other aspect of thedisclosure delineated herein, the topology is altered by substituting,adding or deleting one or more nucleotides of an anchor sequence withinthe anchor sequence-mediated conjunction. For example, the one or morenucleotides substituted, added or deleted may be within at least oneanchor sequence, such as a binding motif for a conjunction nucleatingmolecule.

In some embodiments, the topology is altered by at least one of thefollowing: modulating DNA methylation at one or more sites within theanchor sequence-mediated conjunction; changing an orientation of atleast one common nucleotide sequence, such as a binding motif for aconjunction nucleating molecule; altering a spatial separation withinthe anchor sequence-mediated conjunction; altering a free energy ofrotation within the anchor sequence-mediated conjunction; and altering apositional degree of freedom within the anchor sequence-mediatedconjunction.

In some additional embodiments, the topology is altered by any one ormore of the following: disrupting the anchor sequence-mediatedconjunction, forming a non-naturally occurring anchor sequence-mediatedconjunction, forming a plurality of non-naturally occurring anchorsequence-mediated conjunctions, and introducing an exogenous anchorsequence.

In certain embodiments, the topology is altered to result in amodulation, e.g., stable, of transcription, such as a modulation thatpersists for at least about 1 hr to about 30 days, or at least about 2hrs, 6 hrs, 12 hrs, 18 hrs, 24 hrs, 2 days, 3, days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30days, or longer or any time therebetween.

In certain embodiments, the topology is altered to result in amodulation, e.g., transient, of transcription, such as a modulation thatpersists for no more than about 30 mins to about 7 days, or no more thanabout 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 19hrs, 20 hrs, 21 hrs, 22 hrs, 24 hrs, 36 hrs, 48 hrs, 60 hrs, 72 hrs, 4days, 5 days, 6 days, 7 days, or any time therebetween.

In some embodiments, the method further includes modulating aconjunction nucleating molecule, such as a binding affinity for ananchor sequence within the anchor sequence-mediated conjunction, thatinteracts with the anchor sequence-mediated conjunction.

In certain embodiments, the anchor sequence-mediated conjunctionincludes at least a first anchor sequence and a second anchor sequence.In one embodiment, the anchor sequence-mediated conjunction is mediatedby a first conjunction nucleating molecule bound to the first anchorsequence, a second conjunction nucleating molecule bound to the secondanchor sequence, and an association between the first and secondconjunction nucleating molecules. In another embodiment, the first orsecond conjunction nucleating molecule has a binding affinity for theanchor sequence greater than or less than a reference value, such as abinding affinity for the anchor sequence in the absence of thealteration.

In some embodiments, the second anchor sequence is non-contiguous withthe first anchor sequence. In one embodiment, the anchorsequence-mediated conjunction is mediated by a first conjunctionnucleating molecule bound to the first anchor sequence, a secondconjunction nucleating molecule bound to the non-contiguous secondanchor sequence, and an association between the first and secondconjunction nucleating molecules. In another embodiment, the first orsecond conjunction nucleating molecule has a binding affinity for theanchor sequence greater than or less than a reference value, such as thebinding affinity for the anchor sequence in the absence of thealteration.

In some embodiments where the anchor sequences are non-contiguous withone another, the first anchor sequence is separated from the secondanchor sequence by about 500 bp to about 500 Mb, about 750 bp to about200 Mb, about 1 kb to about 100 Mb, about 25 kb to about 50 Mb, about 50kb to about 1 Mb, about 100 kb to about 750 kb, about 150 kb to about500 kb, or about 175 kb to about 500 kb. In some embodiments, the firstanchor sequence is separated from the second anchor sequence by about500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kb, 5 kb, 10 kb, 15 kb, 20 kb,25 kb, 30 kb, 35 kb, 40 kb, 45 kb, 50 kb, 55 kb, 60 kb, 65 kb, 70 kb, 75kb, 80 kb, 85 kb, 90 kb, 95 kb, 100 kb, 125 kb, 150 kb, 175 kb, 200 kb,225 kb, 250 kb, 275 kb, 300 kb, 350 kb, 400 kb, 500 kb, 600 kb, 700 kb,800 kb, 900 kb, 1 Mb, 2 Mb, 3 Mb, 4 Mb, 5 Mb, 6 Mb, 7 Mb, 8 Mb, 9 Mb, 10Mb, 15 Mb, 20 Mb, 25 Mb, 50 Mb, 75 Mb, 100 Mb, 200 Mb, 300 Mb, 400 Mb,500 Mb, or any size therebetween.

In certain embodiments, the first anchor sequence and second anchorsequence each includes a common nucleotide sequence, such as a bindingmotif for a conjunction nucleating molecule, such as one describedherein. In some embodiments, the first anchor sequence and second anchorsequence include different sequences, such as the first anchor sequencecomprises a binding motif for a conjunction nucleating molecule and thesecond anchor sequence comprises an anchor sequence a binding motif foranother molecule, e.g., another conjunction nucleating molecule.

In some embodiments, the anchor sequence-mediated conjunction includes aplurality of anchor sequences. In one embodiment, at least one of anchorsequences includes a CTCF binding motif.

In some more embodiments, the anchor sequence-mediated conjunctioncomprises a loop, such as an intra-chromosomal loop. In one embodiment,the loop includes a first anchor sequence, a nucleic acid sequence, atranscriptional control sequence, such as an enhancing or silencingsequence, and a second anchor sequence. In another embodiment, the loopincludes, in order, a first anchor sequence, a transcriptional controlsequence, and a second anchor sequence; or a first anchor sequence, anucleic acid sequence, and a second anchor sequence. In yet anotherembodiment, either one or both of the nucleic acid sequence and thetranscriptional control sequence is located within or outside the loop.

In certain embodiments, the anchor sequence-mediated conjunction has aplurality of loops. In one embodiment, the anchor sequence-mediatedconjunction includes the plurality of loops, and the anchorsequence-mediated conjunction includes at least one of an anchorsequence, a nucleic acid sequence, and a transcriptional controlsequence in one or more of the loops.

In some embodiments, transcription of the nucleic acid sequence ismodulated, such as transcription of a target nucleic acid sequence, ascompared with a reference value, e.g., transcription of the targetsequence in the absence of the altered anchor sequence-mediatedconjunction.

In some embodiments, transcription is activated by inclusion of anactivating loop. In one embodiment, the anchor sequence-mediatedconjunction includes a transcriptional control sequence, such as anenhancing sequence, that increases transcription of the nucleic acidsequence. In some more embodiments, transcription is activated byexclusion of a repressive loop. In one embodiment, the anchorsequence-mediated conjunction excludes a transcriptional controlsequence, such as a silencing sequence, that decreases transcription ofthe nucleic acid sequence.

In some embodiments, transcription is repressed by inclusion of arepressive loop. In one embodiment, the anchor sequence-mediatedconjunction includes a transcriptional control sequence such as asilencing sequence, that decreases transcription of the nucleic acidsequence. In some more embodiments, transcription is repressed byexclusion of an activating loop. In one embodiment, the anchorsequence-mediated conjunction excludes a transcriptional controlsequence, such as an enhancing sequence, that increases transcription ofthe nucleic acid sequence.

In certain embodiments, the anchor sequence-mediated conjunction isaltered in vivo, such as in a subject, e.g., a human subject. In someembodiments, the methods delineated herein further include administeringa targeting moiety selected from at least one of an exogenousconjunction nucleating molecule, a nucleic acid encoding the conjunctionnucleating molecule, and a fusion of a sequence targeting polypeptideand a conjunction nucleating molecule to the subject. In one embodiment,the conjunction nucleating molecule disrupts, such as throughcompetitive binding, the binding of an endogenous conjunction nucleatingmolecule to its binding site. In another embodiment, the targetingmoiety includes a sequence targeting polypeptide, such as an enzyme,e.g., Cas9. In yet another embodiment, the targeting moiety furtherincludes a conjunction nucleating molecule. In still another embodiment,the targeting moiety further includes a guide RNA or nucleic acidencoding the guide RNA.

In some embodiments, the administration includes administering a vector,such as a viral vector, e.g., lentiviral vector, that comprises thenucleic acid encoding the targeting moiety, e.g., the conjunctionnucleating molecule. In some more embodiments, the administrationincludes administering a formulation, such as formulated in a polymericcarrier, e.g., a liposome.

In one aspect, the disclosure includes an engineered cell comprising atargeted alteration in an anchor sequence-mediated conjunction.

In another aspect, the disclosure includes an engineered nucleic acidsequence comprising an anchor sequence-mediated conjunction with atargeted alteration.

In various embodiments of the above aspects or any other aspect of thedisclosure delineated herein, the targeted alteration includes any oneor more of the following: a substitution, addition or deletion of one ormore nucleotides of an anchor sequence within the anchorsequence-mediated conjunction; a substitution, addition or deletion ofone or more nucleotides in at least one anchor sequence, e.g., a CTCFbinding motif; an alteration of one or more DNA methylation sites withinthe anchor sequence-mediated conjunction; and at least one exogenousanchor sequence.

In some embodiments, the targeted alteration alters at least oneconjunction nucleating molecule binding site, such as altering itsbinding affinity for the conjunction nucleating molecule. In some moreembodiments, the targeted alteration changes an orientation of at leastone common nucleotide sequence, e.g., a CTCF binding motif; disrupts theanchor sequence-mediated conjunction; and forms a non-naturallyoccurring anchor sequence-mediated conjunction.

In certain embodiments, the anchor sequence-mediated conjunctionincludes at least a first anchor sequence and a second anchor sequence.In one embodiment, the anchor sequence-mediated conjunction is mediatedby a first conjunction nucleating molecule bound to the first anchorsequence, a second conjunction nucleating molecule bound to the secondanchor sequence, and an association between the first and secondconjunction nucleating molecules. In another embodiment, the first orsecond conjunction nucleating molecule has a binding affinity for theanchor sequence greater than or less than a reference value, such as abinding affinity for the anchor sequence in the absence of thealteration.

In some embodiments, the second anchor sequence is non-contiguous withthe first anchor sequence. In one embodiment, the anchorsequence-mediated conjunction is mediated by a first conjunctionnucleating molecule bound to the first anchor sequence, a secondconjunction nucleating molecule bound to the non-contiguous secondanchor sequence, and an association between the first and secondconjunction nucleating molecules. In another embodiment, the first orsecond conjunction nucleating molecule has a binding affinity for theanchor sequence greater than or less than a reference value, such as thebinding affinity for the anchor sequence in the absence of thealteration.

In some embodiments where the anchor sequences are non-contiguous withone another, the first anchor sequence is separated from the secondanchor sequence by about 500 bp to about 500 Mb, about 750 bp to about200 Mb, about 1 kb to about 100 Mb, about 25 kb to about 50 Mb, about 50kb to about 1 Mb, about 100 kb to about 750 kb, about 150 kb to about500 kb, or about 175 kb to about 500 kb. In some embodiments, the firstanchor sequence is separated from the second anchor sequence by about500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kb, 5 kb, 10 kb, 15 kb, 20 kb,25 kb, 30 kb, 35 kb, 40 kb, 45 kb, 50 kb, 55 kb, 60 kb, 65 kb, 70 kb, 75kb, 80 kb, 85 kb, 90 kb, 95 kb, 100 kb, 125 kb, 150 kb, 175 kb, 200 kb,225 kb, 250 kb, 275 kb, 300 kb, 350 kb, 400 kb, 500 kb, 600 kb, 700 kb,800 kb, 900 kb, 1 Mb, 2 Mb, 3 Mb, 4 Mb, 5 Mb, 6 Mb, 7 Mb, 8 Mb, 9 Mb, 10Mb, 15 Mb, 20 Mb, 25 Mb, 50 Mb, 75 Mb, 100 Mb, 200 Mb, 300 Mb, 400 Mb,500 Mb, or any size therebetween.

In certain embodiments, the first anchor sequence and second anchorsequence each includes a common nucleotide sequence, such as a CTCFbinding motif. In some embodiments, the first anchor sequence and secondanchor sequence include different sequences, such as the first anchorsequence comprises a CTCF binding motif and the second anchor sequencecomprises an anchor sequence other than a CTCF binding motif.

In some embodiments, the anchor sequence-mediated conjunction includes aplurality of anchor sequences. In one embodiment, at least one of anchorsequences includes a CTCF binding motif.

In some more embodiments, the anchor sequence-mediated conjunctioncomprises a loop, such as an intra-chromosomal loop. In one embodiment,the loop includes a first anchor sequence, a nucleic acid sequence, atranscriptional control sequence, such as an enhancing or silencingsequence, and a second anchor sequence. In another embodiment, the loopincludes, in order, a first anchor sequence, a transcriptional controlsequence, and a second anchor sequence; or a first anchor sequence, anucleic acid sequence, and a second anchor sequence. In yet anotherembodiment, either one or both of the nucleic acid sequence and thetranscriptional control sequence is located within or outside the loop.

In certain embodiments, the anchor sequence-mediated conjunction has aplurality of loops. In one embodiment, the anchor sequence-mediatedconjunction includes the plurality of loops, and the anchorsequence-mediated conjunction includes at least one of an anchorsequence, a nucleic acid sequence, and a transcriptional controlsequence in one or more of the loops.

In some embodiments, transcription of the nucleic acid sequence ismodulated, such as transcription of a target nucleic acid sequence, ascompared with a reference value, e.g., transcription of the targetsequence in the absence of the altered anchor sequence-mediatedconjunction.

In some embodiments, transcription is activated by inclusion of anactivating loop. In one embodiment, the anchor sequence-mediatedconjunction includes a transcriptional control sequence, such as anenhancing sequence, that increases transcription of the nucleic acidsequence. In some more embodiments, transcription is activated byexclusion of a repressive loop. In one embodiment, the anchorsequence-mediated conjunction excludes a transcriptional controlsequence, such as a silencing sequence, that decreases transcription ofthe nucleic acid sequence.

In some embodiments, transcription is repressed by inclusion of arepressive loop. In one embodiment, the anchor sequence-mediatedconjunction includes a transcriptional control sequence such as asilencing sequence, that decreases transcription of the nucleic acidsequence. In some more embodiments, transcription is repressed byexclusion of an activating loop. In one embodiment, the anchorsequence-mediated conjunction excludes a transcriptional controlsequence, such as an enhancing sequence, that increases transcription ofthe nucleic acid sequence.

In some embodiments, the disclosure includes a pharmaceuticalcomposition with the engineered cell described herein, or the engineerednucleic acid sequence described herein. In some more embodiments, thedisclosure includes a plurality of cells with the engineered celldescribed herein. In some additional embodiments, the disclosureincludes a vector with the engineered nucleic acid sequence describedherein.

In one aspect, the disclosure includes a method of treating a disease orcondition comprising administering a targeting moiety selected from atleast one of an exogenous conjunction nucleating molecule, a nucleicacid encoding the conjunction nucleating molecule, and a fusion of asequence targeting polypeptide and a conjunction nucleating molecule toa subject.

In certain embodiments, the conjunction nucleating molecule disrupts,such as through competitive binding, the binding of an endogenousconjunction nucleating molecule to its binding site.

In some embodiments, the targeting moiety includes a sequence targetingpolypeptide, such as an enzyme, e.g., Cas9. In some embodiments, thetargeting moiety further includes a conjunction nucleating molecule. Insome more embodiments, the targeting moiety further includes a guide RNAor nucleic acid encoding the guide RNA. In some additional embodiments,the targeting moiety targets one or more nucleotides, such as throughCRISPR, TALEN, dCas9, recombination, transposon, etc., of an anchorsequence within the anchor sequence-mediated conjunction forsubstitution, addition or deletion. In some embodiments, the targetingmoiety targets one or more DNA methylation sites within the anchorsequence-mediated conjunction. In some more embodiments, the targetingmoiety introduces at least one of the following: at least one exogenousanchor sequence; an alteration in at least one conjunction nucleatingmolecule binding site, such as by altering binding affinity for theconjunction nucleating molecule; a change in an orientation of at leastone common nucleotide sequence, such as a CTCF binding motif; and asubstitution, addition or deletion in at least one anchor sequence, suchas a CTCF binding motif.

In certain embodiments, the administration includes administering avector, e.g., a viral vector, that comprises the nucleic acid encodingthe targeting moiety, e.g., the conjunction nucleating molecule. In somemore embodiments, the administration includes administering aformulation, e.g., a liposome.

In some embodiments, the disease or condition is selected from the groupconsisting of cancer, trinucleotide repeats (Huntington's Chorea,Fragile X, all the spinocerebellar ataxias, Friedrich ataxia, myotonicdystrophy and others), an autosomal dominant condition, a disease of animprinted gene (Prader Willi Syndrome, Angelman Syndrome), a disease ofhaploinsufficiency, a dominant negative mutation (Severe congenitalneutropenia), a viral disease (HIV, HBV, HCV, HPV etc), and anenvironmentally driven transcriptional-epigenetic alteration (effectsfrom smoking, maternal diet on gene expression).

In one aspect, the disclosure includes a pharmaceutical compositioncomprising at least one polypeptide, e.g., a membrane translocatingpolypeptide, with each comprising at least one sequence of ABX^(n)C,where A is selected from a hydrophobic amino acid or an amide containingbackbone, e.g., aminoethyl-glycine, with a nucleic acid side chain; Band C may be the same or different, and are each independently selectedfrom arginine, asparagine, glutamine, lysine, and analogs thereof; X iseach independently a hydrophobic amino acid or X is each independentlyan amide containing backbone, e.g., aminoethyl-glycine, with a nucleicacid side chain; and n is an integer from 1 to 4, wherein thepolypeptide is capable of hybridizing a nucleic acid sequence within ananchor sequence-mediated conjunction (e.g., anchor sequence of an anchorsequence-mediated conjunction, e.g., CTCF binding motif, BORIS bindingmotif, cohesin binding motif, USF1 binding motif, YY1 binding motif,TATA-box, ZNF143 binding motif, etc).

The composition as described in various embodiments of the above aspectmay be utilized in any other aspect delineated herein. In someembodiments, the targeting moiety of one or more embodiments describedherein comprises a membrane translocating polypeptide, e.g., thepolypeptide described herein.

In some embodiments, the hydrophobic amino acid is selected fromalanine, valine, isoleucine, leucine, methionine, phenylalanine,tyrosine, trytophan, and analogs thereof. In some embodiments, B isselected from arginine or glutamine. In some embodiments, C is arginine.In some embodiments, n is 2.

In some embodiments, the polypeptides have sizes in the range of about 5to about 50 amino acid units in length.

In some embodiments, the composition comprises two or more polypeptidesthat are linked to one another. In some embodiments, the polypeptidesare linked to one another, e.g., amino acids on one polypeptide arelinked with one or more amino acids or a carboxy or amino terminal onanother polypeptide, branched polypeptide, or through new peptide bonds,linear polypeptide. In some embodiments, the polypeptides are linked bya linker as described herein.

In some embodiments, the nucleic acid side chain is independentlyselected from the group consisting of a purine side chain, a pyrimidineside chain, and a nucleic acid analog side chain. In some embodiments,the nucleic acid side chain hybridizes to the heterologous moiety,wherein the heterologous moiety comprises a nucleic acid side chain,e.g., a PNA, or nucleic acid.

In some embodiments, the composition comprises the membranetranslocating polypeptide and at least one heterologous moiety. In oneembodiment, the heterologous moiety is a conjunction nucleating moleculethat interacts with the anchor sequence-mediated conjunction. In anotherembodiment, the heterologous moiety is a sequence targeting polypeptide,e.g. Cas9. In another embodiment, the heterologous moiety is a guide RNAor nucleic acid encoding the guide RNA.

In some embodiments, the heterologous moiety is selected from the groupconsisting of a small molecule (e.g., a drug), a peptide (e.g., ligand),and a nucleic acid (e.g., siRNA, DNA, modified RNA, RNA). In anotherembodiment, the heterologous moiety possesses at least one effectoractivity selected from the group consisting of modulates a biologicalactivity, binds a regulatory protein, modulates enzymatic activity,modulates substrate binding, modulates receptor activation, modulatesprotein stability/degradation, and modulates transcriptstability/degradation. In another embodiment, the heterologous moietypossesses at least one targeted function selected from the groupconsisting of modulates a function, modulates a molecule (e.g., enzyme,protein or nucleic acid), and is localized to a specific location. Inanother embodiment, the heterologous moiety is a tag or label, e.g.,cleavable. In another embodiment, the heterologous moiety is selectedfrom the group consisting of an epigenetic modifying agent, epigeneticenzyme, a bicyclic peptide, a transcription factor, a DNA or proteinmodification enzyme, a DNA-intercalating agent, an efflux pumpinhibitor, a nuclear receptor activator or inhibitor, a proteasomeinhibitor, a competitive inhibitor for an enzyme, a protein synthesisinhibitor, a nuclease, a protein fragment or domain, a tag or marker, anantigen, an antibody or antibody fragment, a ligand or a receptor, asynthetic or analog peptide from a naturally-bioactive peptide, ananti-microbial peptide, a pore-forming peptide, a targeting or cytotoxicpeptide, a degradation or self-destruction peptide, a CRISPR system orcomponent thereof, DNA, RNA, artificial nucleic acids, a nanoparticle,an oligonucleotide aptamer, a peptide aptamer, and an agent with poorpharmacokinetics or pharmacodynamics (PK/PD).

In some embodiments, the composition further comprises two or moreheterologous moieties linked, e.g., via a linker or directly, to thepolypeptide on amino termini, on carboxy termini, all termini, acombination of some carboxy and some amino termini of the polypeptides,one or more amino acids of the polypeptide, or any combination thereof.In some embodiments, the heterologous moiety is linked, e.g., via alinker or directly, to one of the polypeptides on an amino terminus, acarboxy terminus, both termini, or one or more amino acids of thepolypeptide.

In some embodiments, the composition further comprises a linker, e.g.,between polypeptides or between the polypeptide and the heterologousmoiety. The linker may be a chemical bond, e.g., one or more covalentbonds or non-covalent bonds. In some embodiments, the linker is apeptide linker (e.g., a non ABX^(n)C polypeptide). Such a linker may bebetween 2-30 amino acids, or longer. The linker includes flexible, rigidor cleavable linkers described herein.

In some embodiments, the composition modulates DNA methylation at one ormore sites within the anchor sequence-mediated conjunction.

In some embodiments, the composition transiently modulatestranscription, e.g., a modulation that persists for no more than about30 mins to about 7 days, or no more than about 1 hr, 2 hrs, 3 hrs, 4hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs,14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 19 hrs, 20 hrs, 21 hrs, 22 hrs,24 hrs, 36 hrs, 48 hrs, 60 hrs, 72 hrs, 4 days, 5 days, 6 days, 7 days,or any time therebetween.

In some embodiments, the composition stably modulates transcription,e.g., a modulation that persists for at least about 1 hr to about 30days, or at least about 2 hrs, 6 hrs, 12 hrs, 18 hrs, 24 hrs, 2 days, 3,days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days,12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days,20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days,28 days, 29 days, 30 days, or longer or any time therebetween.

In some embodiments, the composition modulates a conjunction nucleatingmolecule, e.g. a binding affinity for an anchor sequence within theanchor sequence-mediated conjunction, that interacts with the anchorsequence-mediated conjunction.

In some embodiments, the composition disrupts, e.g., by competitivebinding, binding of an endogenous conjunction nucleating molecule to itsbinding site.

In one aspect, the disclosure includes a method of modifying expressionof a target gene, comprising altering an anchor sequence-mediatedconjunction associated with the target gene, wherein the alterationmodulates transcription of the target gene.

In one aspect, the disclosure includes a method of modifying expressionof a target gene, comprising administering the composition describedherein to a cell, tissue or subject.

In one aspect, the disclosure includes a method of modulatingtranscription of a nucleic acid sequence comprising administering thecomposition described herein to alter an anchor sequence-mediatedconjunction, e.g., a loop, that modulates transcription of a nucleicacid sequence, wherein the altered anchor sequence-mediated conjunctionmodulates transcription of the nucleic acid sequence.

The method as described in various embodiments of the above aspect maybe utilized in any other aspect delineated herein.

In some embodiments, the composition modulates DNA methylation at one ormore sites within the anchor sequence-mediated conjunction.

In some embodiments, altering the anchor sequence-mediated conjunctionresults in a transient modulation of transcription, e.g., a modulationthat persists for no more than about 30 mins to about 7 days, or no morethan about 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs,10 hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs,19 hrs, 20 hrs, 21 hrs, 22 hrs, 24 hrs, 36 hrs, 48 hrs, 60 hrs, 72 hrs,4 days, 5 days, 6 days, 7 days, or any time therebetween.

In some embodiments, altering the anchor sequence-mediated conjunctionresults in a stable modulation of transcription, e.g., a modulation thatpersists for at least about 1 hr to about 30 days, or at least about 2hrs, 6 hrs, 12 hrs, 18 hrs, 24 hrs, 2 days, 3, days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30days, or longer or any time therebetween.

In some embodiments, the composition modulates a conjunction nucleatingmolecule, e.g. a binding affinity for an anchor sequence within theanchor sequence-mediated conjunction, that interacts with the anchorsequence-mediated conjunction.

In some embodiments, the composition disrupts, e.g., by competitivebinding, binding of an endogenous conjunction nucleating molecule to itsbinding site.

In some embodiments, the heterologous moiety is a sequence targetingpolypeptide, e.g. Cas9. In some embodiments, the heterologous moiety isa guide RNA or nucleic acid encoding the guide RNA.

In one aspect, the disclosure includes a method of modulating geneexpression comprising providing the composition described herein, e.g.,the heterologous moiety inhibits CpG binding, is an endogenous effector,is an exogenous effector, or agonist or antagonist thereof.

In one aspect, the disclosure includes a method of delivering atherapeutic comprising administering the composition described herein toa subject, wherein the heterologous moiety is the therapeutic, andwherein the composition increases intracellular delivery of thetherapeutic as compared to the therapeutic alone.

The method as described in various embodiments of the above aspect maybe utilized in any other aspect delineated herein.

In some embodiments, the composition is targeted to a specific cell, ora specific tissue. For example, the composition is targeted to anepithelial, connective, muscular, or nervous tissue or cells, orcombinations thereof. For example, the composition is targeted to a cellor tissue of a particular organ system, e.g., the cardiovascular system(heart, vasculature); digestive system (esophagus, stomach, liver,gallbladder, pancreas, intestines, colon, rectum and anus); endocrinesystem (hypothalamus, pituitary gland, pineal body or pineal gland,thyroid, parathyroids, adrenal glands); excretory system (kidneys,ureters, bladder); lymphatic system (lymph, lymph nodes, lymph vessels,tonsils, adenoids, thymus, spleen); integumentary system (skin, hair,nails); muscular system (e.g., skeletal muscle); nervous system (brain,spinal cord, nerves); reproductive system (ovaries, uterus, mammaryglands, testes, vas deferens, seminal vesicles, prostate); respiratorysystem (pharynx, larynx, trachea, bronchi, lungs, diaphragm); skeletalsystem (bone, cartilage), and combinations thereof. In some embodiments,the composition crosses a blood-brain-barrier, a placental membrane, ora blood-testis barrier.

In some embodiments, the composition is administered systemically. Insome embodiments, the administration is non-parenteral and thetherapeutic is a parenteral therapeutic.

In some embodiments, the composition has improved PK/PD, e.g., increasedpharmacokinetics or pharmacodynamics, such as improved targeting,absorption, or transport (e.g., at least 5%, 10%, 15%, 20%, 30%, 40%,50%, 60%, 75%, 80%, 90% improved or more) as compared to the therapeuticalone. In some embodiments, the composition has reduced undesirableeffects, such as reduced diffusion to non-target location, off-targetactivity, or toxic metabolism, as compared to the therapeutic alone(e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% ormore reduced, as compared to the therapeutic alone). In someembodiments, the composition increases efficacy and/or decreasestoxicity of the therapeutic (e.g., at least 5%, 10%, 15%, 20%, 30%, 40%,50%, 60%, 75%, 80%, 90% or more) as compared to the therapeutic alone.

In one aspect, the disclosure includes a method of intracellulardelivery of a therapeutic comprising contacting a cell with thecomposition described herein, wherein the heterologous moiety is thetherapeutic, and wherein the composition increases intracellulardelivery of the therapeutic as compared to the therapeutic alone.

The method as described in various embodiments of the above aspect maybe utilized in any other aspect delineated herein.

In some embodiments, the composition has differential PK/PD as comparedto the therapeutic alone. For example, the composition exhibitsincreased or decreased absorption or distribution, metabolism orexcretion (e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%,80%, 90% or more increased or decreased), as compared to the therapeuticalone.

In some embodiments, the composition is administered at a dosesufficient to increase intracellular delivery of the therapeutic withoutsignificantly increasing endocytosis, e.g., less than about 50%, 40%,20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or any percentage therebetween. Insome embodiments, the composition is administered at a dose sufficientto increase intracellular delivery of the therapeutic withoutsignificantly increasing calcium influx, e.g., less than about 50%, 40%,20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or any percentage therebetween. Insome embodiments, the composition is administered at a dose sufficientto increase intracellular delivery of the therapeutic withoutsignificantly increasing endosomal activity, e.g., less than about 50%,40%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or any percentage therebetween.

In one aspect, the disclosure includes a method of modulatingtranscription of a gene in a cell comprising contacting the cell withthe composition described herein, wherein the composition targets thegene and modulates its transcription.

The method as described in various embodiments of the above aspect maybe utilized in any other aspect delineated herein.

In some embodiments, the composition is administered in an amount andfor a time sufficient to effect intracellular delivery of thetherapeutic with decreased off target transcriptional activity comparedto the heterologous moiety alone, e.g., without significantly alteringoff-target transcriptional activity.

In one aspect, the disclosure includes a method of modulating a membraneprotein, e.g., such as an ion channel, a cell surface receptor and asynaptic receptor, on a cell comprising contacting the cell with thecomposition described herein, wherein the composition targets the celland modulates the membrane protein.

In one aspect, the disclosure includes a method of inducing cell deathcomprising contacting a cell with the composition described herein,wherein the composition targets the cell and induces apoptosis.

The method as described in various embodiments of the above aspect maybe utilized in any other aspect delineated herein.

In some embodiments, the composition targets a cell harboring a viralDNA sequence or a mutation in a gene. In one embodiment, the cell isvirally infected. In another embodiment, the cell harbors a geneticmutation. In some embodiments, the composition targets a cell in theearly stages of necrosis, e.g., binding the necrotic cell marker.

In one aspect, the disclosure includes a method of increasingbioavailability of a therapeutic comprising administering thecomposition described herein, wherein the therapeutic is theheterologous moiety.

The method as described in various embodiments of the above aspect maybe utilized in any other aspect delineated herein.

In some embodiments, the composition improves (e.g., by at least 5%,10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more) at least onePK/PD parameter, such as improved targeting, absorption, or transport,as compared to the therapeutic alone. In some embodiments, thecomposition reduces (e.g., by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%,60%, 75%, 80%, 90% or more) at least one unwanted parameter, such asreduced diffusion to non-target location, off-target activity, or toxicmetabolism, as compared to the therapeutic alone. In some embodiments,the composition increases efficacy and/or decreases toxicity of thetherapeutic as compared to the therapeutic alone.

In one aspect, the disclosure includes a method of treating an acute orchronic infection comprising administering the composition describedherein.

The method as described in various embodiments of the above aspect maybe utilized in any other aspect delineated herein.

In some embodiments, the composition targets an infected cell harboringa pathogen. In some embodiments, the infection is caused by a pathogenselected from the group consisting of a virus, bacteria, parasite, and aprion. In some embodiments, the composition induces cell death in theinfected cell, e.g., the heterologous moiety is an antibacterial, anantiviral, or an antiparasitic therapeutic.

In one aspect, the disclosure includes a method of treating a cancercomprising administering the composition described herein.

The method as described in various embodiments of the above aspect maybe utilized in any other aspect delineated herein.

In some embodiments, the heterologous moiety is a therapeutic thatmodulates gene expression of one or more genes.

In some embodiments, the composition targets a cancer cell harboring amutation in a gene. In some embodiments, the composition induces celldeath in the cancer cell, e.g., the heterologous moiety is achemotherapeutic agent.

In one aspect, the disclosure includes a method of treating aneurological disease or disorder comprising administering thecomposition described herein.

The method as described in various embodiments of the above aspect maybe utilized in any other aspect delineated herein.

In some embodiments, the composition modulates neuroreceptor activity oractivation of a neurotransmitter, neuropeptide, or neuroreceptor.

In some embodiments, the neurological disease or disorder is Dravet'ssyndrome.

In one aspect, the disclosure includes a method of treating adisease/disorder/condition in a subject comprising administering thecomposition described herein, wherein the composition modulatestranscription to treat the disease/disorder/condition.

The method as described in various embodiments of the above aspect maybe utilized in any other aspect delineated herein.

In some embodiments, the disease/disorder/condition is a geneticdisease.

In one aspect, the disclosure includes a method of inducing immunetolerance comprising providing the composition described herein, e.g.,the heterologous moiety is an antigen.

In one aspect, the disclosure includes a method of altering expressionof a target gene in a genome, comprising: administering to the genome apharmaceutical composition comprising (a) a targeting moiety and (b) aDNA sequence comprising an anchor sequence, wherein the anchor sequencepromotes the formation of a conjunction that brings a gene expressionfactor (an enhancing sequence, a silencing/repressive sequence) intooperable linkage with the target gene.

In one aspect, the disclosure includes a system for pharmaceutical usecomprising a protein comprising a first polypeptide domain thatcomprises a Cas or modified Cas protein and a second polypeptide domainthat comprises a polypeptide having DNA methyltransferase activity [orassociated with demethylation or deaminase activity], in combinationwith at least one guide RNA (gRNA) or antisense DNA oligonucleotide thattargets the protein to an anchor sequence of a target anchorsequence-mediated conjunction, wherein the system is effective to alter,in a human cell, the target anchor sequence-mediated conjunction.

In one aspect, the disclosure includes a system for altering, in a humancell, expression of a target gene, comprising a targeting moiety (e.g.,a gRNA, an LDB) that associates with an anchor sequence associated withthe target gene, optionally, a heterologous moiety (e.g., an enzyme,e.g., a nuclease or deactivated nuclease (e.g., a Cas9, dCas9), amethylase, a de-methylase, a deaminase) operably linked to the targetingmoiety, wherein the system is effective to modulate a conjunctionmediated by the anchor sequence and alter expression of the target gene.

The system as described in various embodiments of the above aspect maybe utilized in any other aspect delineated herein.

In some embodiments, the targeting moiety and the effector moiety arelinked. In some embodiments, the system comprises a syntheticpolypeptide comprising the targeting moiety and the heterologous moiety.In some embodiments, the system comprises a nucleic acid vector orvectors encoding at least one of the targeting moiety and theheterologous moiety.

The aspects as described here may be utilized with any one or more ofthe embodiments delineated herein.

Definitions

The term “anchor sequence” as used herein, refers to a sequencerecognized by a conjunction nucleating agent (e.g., a nucleatingprotein) that binds sufficiently to form an anchor sequence-mediatedconjunction, e.g., a loop. In some embodiments, an anchor sequencecomprises one or more CTCF binding motifs. In some embodiments, ananchor sequence is not located within a gene coding region. In someembodiments, an anchor sequence is located within an intergenic region.In some embodiments, an anchor sequence is not located within either ofan enhancer or a promoter. In some embodiments, an anchor sequence islocated at least 400 bp, at least 450 bp, at least 500 bp, at least 550bp, at least 600 bp, at least 650 bp, at least 700 bp, at least 750 bp,at least 800 bp, at least 850 bp, at least 900 bp, at least 950 bp, orat least 1 kb away from any transcription start site. In someembodiments, an anchor sequence is located within a region that is notassociated with genomic imprinting, monoallelic expression, and/ormonoallelic epigenetic marks. In some embodiments of the presentdisclosure, technologies are provided that may specifically target aparticular anchor sequence or anchor sequences, without targeting otheranchor sequences (e.g., sequences that may contain a conjunctionnucleating agent (e.g., CTCF) binding motif in a different context);such targeted anchor sequences may be referred to as the “target anchorsequence”. In some embodiments, sequence and/or activity of a targetanchor sequence is modulated while sequence and/or activity of one ormore other anchor sequences that may be present in the same system(e.g., in the same cell and/or in some embodiments on the same nucleicacid molecule—e.g., the same chromosome) as the targeted anchor sequenceis not modulated.

The phrase “anchor sequence-mediated conjunction” as used herein, refersto a DNA structure, in some cases, a loop, that occurs and/or ismaintained via the physical interaction or binding of at least twoanchor sequences in the DNA by one or more proteins, such as nucleatingproteins, or one or more proteins and/or a nucleic acid entity (such asRNA or DNA), that bind the anchor sequences to enable spatial proximityand functional linkage between the anchor sequences (see FIG. 1).

The term “associated with” as used herein, refers to a target gene isassociated with an anchor sequence-mediated conjunction if the formationor disruption of the anchor sequence-mediated conjunction causes analteration in expression (e.g., transcription) of the gene. For example,the formation or disruption of the anchor sequence-mediated conjunctioncauses an enhancing or silencing/repressive sequence to associate withor become unassociated with the gene.

The phrase “non-naturally occurring anchor sequence-mediatedconjunction” as used herein, refers the formation of an anchorsequence-mediated conjunction not existing in nature. The generation ofthe non-naturally occurring anchor sequence-mediated conjunction may bethrough, but not limited to, alteration, addition or deletion of one ormore anchor sequences, and alteration of one or more conjunctionnucleating molecules.

The term “common nucleotide sequence” as used herein, refers to aconjunction nucleating molecule binding site in an anchor sequence.Examples of common nucleotide sequences include, but are not limited to,CTCF binding motifs, USF1 binding motifs, YY1 binding motifs, TAF3binding motifs, and ZNF143 binding motifs.

By the term “conjunction nucleating agent” as used herein, refers to aprotein that associates with an anchor sequence directly or indirectlyand may interact with one or more conjunction nucleating agents (thatmay interact with an anchor sequence or other nucleic acids) to form adimer (or higher order structure) comprised of two or more suchconjunction nucleating agents, which may or may not be identical to oneanother. When conjunction nucleating agents associated with differentanchor sequences associate with each other so that the different anchorsequences are maintained in physical proximity with one another, thestructure generated thereby is an anchor-sequence-mediated conjunction.That is, the close physical proximity of a conjunction nucleatingmolecule-anchor sequence interacting with another conjunction nucleatingmolecule-anchor sequence generates an anchor sequence-mediatedconjunction (e.g., in some cases, a DNA loop), that begins and ends atthe anchor sequence (see FIG. 2). As those skilled in the art, readingthe present specification will immediately appreciate, terms such as“nucleating polypeptide”, “nucleating molecule”, “conjunction nucleatingprotein”, may sometimes be used to refer to a conjunction nucleatingagent. As will similarly be immediately appreciated by those skilled inthe art reading the present specification, an assembles collection oftwo or more conjunction nucleating agents (which may, in someembodiments, include multiple copies of the same agent and/or in someembodiments one or more of each of a plurality of different agents) maybe referred to as a “complex”, a “dimer” a “multimer”, etc.

The term “loop” refers to a type of chromatin structure that may becreated by co-localization of two or more anchor sequences as an anchorsequence-mediated conjunction. Thus, the loop is formed as a consequenceof the interaction of at least two anchor sequences in DNA with one ormore proteins, such as nucleating proteins, or one or more proteinsand/or a nucleic acid entity (such as RNA or DNA), that bind the anchorsequences to enable spatial proximity and functional linkage between theanchor sequences. Those skilled in the art, reading the presentspecification, will appreciate that a 2D representation of such astructure may be presented as a loop, e.g., as depicted in FIG. 2. An“activating loop” is a structure that is open to active genetranscription, for example, a structure comprising a transcriptioncontrol sequence (enhancing sequence) that enhances transcription. A“repressive loop” is a structure that is closed off from active genetranscription, for example, a structure comprising a transcriptioncontrol sequence (silencing sequence) that represses transcription.

The term “sequence targeting polypeptide” as used herein, refers to aprotein, such as an enzyme, e.g., Cas9, that recognizes or specificallybinds to a target sequence. In some embodiments, the sequence targetingpolypeptide is a catalytically inactive protein, such as dCas9, thatlacks endonuclease activity.

The term “subject,” as used herein refers to an organism, for example, amammal (e.g., a human, a non-human mammal, a non-human primate, aprimate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, acat, or a a dog). In some embodiments a human subject is an adult,adolescent, or pediatric subject. In some embodiments, a subject had adisease or a condition. In some embodiments, the subject is sufferingfrom a disease, disorder or condition, e.g., a disease, disorder orcondition that can be treated as provided herein. In some embodiments, asubject is susceptible to a disease, disorder, or condition; in someembodiments, a susceptible subject is predisposed to and/or shows anincreased risk (as compared to the average risk observed in a referencesubject or population) of developing the disease, disorder or condition.In some embodiments, a subject displays one or more symptoms of adisease, disorder or condition. In some embodiments, a subject does notdisplay a particular symptom (e.g., clinical manifestation of disease)or characteristic of a disease, disorder, or condition. In someembodiments, a subject does not display any symptom or characteristic ofa disease, disorder, or condition. In some embodiments, a subject is apatient. In some embodiments, a subject is an individual to whomdiagnosis and/or therapy is and/or has been administered.

The term “targeting moiety” or “targeting element” as used herein,refers to molecule that specifically binds a sequence in or around theanchor sequence-mediated conjunction. Examples of a targeting moietyinclude, but are not limited to, a sequence targeting polypeptide, suchas an enzyme, e.g., Cas9, a fusion of a sequence targeting polypeptideand a conjunction nucleating molecule, e.g. a fusion of dCas9 and aconjunction nucleating molecule, or a guide RNA or nucleic acid, such asRNA, DNA, or modified RNA or DNA.

The term “transcriptional control sequence” as used herein, refers to anucleic acid sequence that increases or decreases transcription of agene. An “enhancing sequence” increases the likelihood of genetranscription. A “silencing or repressive sequence” decreases thelikelihood of gene transcription. Enhancing and silencing sequences arearound 50-3500 bp in length and may influence gene transcription up to 1Mb away.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of the embodiments of the disclosurewill be better understood when read in conjunction with the appendeddrawings. For the purpose of illustrating the disclosure, there areshown in the drawings embodiments, which are presently exemplified. Itshould be understood, however, that the disclosure is not limited to theprecise arrangement and instrumentalities of the embodiments shown inthe drawings.

FIG. 1 is an illustration depicting the physical interaction or bindingof one conjunction nucleating molecule-anchor sequence with anotherconjunction nucleating molecule-anchor sequence to generate an anchorsequence-mediated conjunction.

FIG. 2 is an illustration depicting methods of targeted disruption andgeneration of anchor sequence-mediated conjunctions, e.g., loops.

FIG. 3 is an illustration depicting one embodiment of modulating geneexpression through the generation of a non-naturally occurring anchorsequence-mediated conjunction (loop inclusion).

FIG. 4 is an illustration depicting methods of modulating geneexpression. The left side of the figure is the same illustration asshown in FIG. 1. The right side of the figure is the disruption of ananchor sequence-mediated conjunction (loop exclusion).

FIG. 5 is an illustration depicting another embodiment of modulatinggene expression through the generation of a non-naturally occurringanchor sequence-mediated conjunction by incorporating a new anchorsequence.

FIG. 6 is an illustration depicting some of the types of anchorsequence-mediated conjunctions.

FIG. 7 illustrates disruption of anchor sequence-mediated conjunctionsupstream of the MYC gene, leading to downregulation of MYC expressionlevels. As further described in Examples 1 and 2, panels A, B, C, and Dillustrates reduction in MYC expression, and panel E depicts a map ofgRNA sequences.

FIG. 8 illustrates disruption of an anchor sequence-mediated conjunctionassociated with the FOXJ3 gene, leading to downregulation of FOXJ3expression levels. As further described in Example 3, panel A depicts amap of gRNA and SNA sequences, and panels B, C, D, and E illustratereduction in FOXJ3 levels.

FIG. 9 illustrates disruption of anchor sequence-mediated conjunctionsassociated with the TUSC5 gene, leading to upregulation of TUSC5expression levels. As further described in Example 4, panel A depictsupregulation of TUSC5 expression levels, and panel B depicts a map ofgRNA sequences.

FIG. 10 illustrates disruption of an anchor sequence-mediatedconjunction upstream of the DAND5 gene, leading to upregulation of DAND5expression levels. As further described in Example 5, panel A depictsupregulation of DAND5 expression levels, and panel B depicts a map ofgRNA sequences.

FIG. 11 illustrated disruption of anchor sequence-mediated conjunctionsupstream or downstream of the SHMT2 gene, leading to downregulation ofSHMT2 expression levels. As further described in Example 6, panels B andC depict maps of gRNA sequences, and panels A and D depictdownregulation of SHMT2 expression levels.

FIG. 12 illustrates disruption of an anchor sequence-mediatedconjunction upstream of the TTC21B gene, leading to upregulation ofTTC21B expression levels. As further described in Example 7, panels Aand B depict upregulation of TTC21B expression levels, and panel Cdepicts a map of gRNA sequences.

FIG. 13 illustrates disruption of an anchor sequence-mediatedconjunction downstream of the CDK6 Gene, leading to downregulation ofCDK6 expression levels. As further described in Example 13, panel Adepicts downregulation of CDK6 expression levels, and panel B depicts amap of gRNA sequences.

FIG. 14 is an illustration of a polypeptide beta hybridized to a CTCFsite in the miR290 loop to physically interfere (mediated by thepolypeptide backbone and the polynucleotide sequence) with the loopingfunction of CTCF.

FIG. 15 is an illustration of multimerized polypeptide beta hybridizedto the promoter of the ELANE gene.

FIG. 16 is an illustration of a polypeptide beta linked to a doublestranded, unmethylated CTCF anchor sequence with specificity for theH19-IGF2 locus to mimic an unmethylated CTCF binding motif on one of thepaternal alleles to form a maternal type of loop.

FIG. 17 provides a summary of certain experimental data for targeteddisruption anchor sequence-mediation conjunctions.

DETAILED DESCRIPTION

The compositions described herein alter a two-dimensional chromatinstructure (e.g., anchor sequence-mediated conjunctions which, as will beappreciated by those skilled in the art, can be graphically representedin two dimensions as having higher order structure than a straight line)in order to modulate gene expression in a subject, e.g., by modifyinganchor sequence-mediated conjunctions in DNA, e.g., genomic DNA.

In one aspect, the disclosure includes a composition comprising atargeting moiety that binds a specific anchor sequence-mediatedconjunction to alter a topology of the anchor sequence-mediatedconjunction, e.g., an anchor sequence-mediated conjunction having aphysical interaction of two or more DNA loci bound by a conjunctionnucleating molecule.

The formation of an anchor sequence-mediated conjunction forces geneexpression regulators to interact with a target gene or spatiallyconstrains the activity of the regulators. Altering anchorsequence-mediated conjunctions allows for gene therapy, e.g., modulatinggene expression, without altering coding sequences of the gene beingmodulated.

In some embodiments, the composition modulates transcription of a geneassociated with an anchor sequence-mediated conjunction by physicallyinterfering between one or more anchor sequences and a conjunctionnucleating molecule. For example, a DNA binding small molecule (e.g.,minor or major groove binders), peptide (e.g., zinc finger, TALEN, novelor modified peptide), protein (e.g., CTCF, modified CTCF with impairedCTCF binding and/or cohesion binding affinity), or nucleic acids (e.g.,ssDNA, modified DNA or RNA, peptide oligonucleotide conjugates, lockednucleic acids, bridged nucleic acids, polyamides, and/or triplex formingoligonucleotides) may physically prevent a conjunction nucleatingmolecule from interacting with one or more anchor sequences to modulategene expression.

In some embodiments, the composition modulates transcription of a geneassociated with an anchor sequence-mediated conjunction by modificationof an anchor sequence, e.g., epigenetic modifications. For example, oneor more anchor sequences associated with an anchor sequence-mediatedconjunction comprising a target gene may be targeted for methylationmodification by a DNA methyltransferase, e.g., dCas9-methyltransferasefusion, e.g., antisense oligonucleotide-enzyme fusion, to modulateexpression of the gene.

In some embodiments, the composition modulates transcription of a geneassociated with an anchor sequence-mediated conjunction by modificationof an anchor sequence, e.g., genomic modifications. For example, one ormore anchor sequences associated with an anchor sequence-mediatedconjunction comprising a target gene may be targeted by a deaminatingenzyme (e.g., deaminating oligonucleotide (e.g. oligo-sodium bisulfateconjugate), dCas-enzyme fusion, antisense oligonucleotide-enzyme fusion,deaminating antisense oligonucleotide-enzyme fusion) to modulateexpression of the gene.

In some embodiments, the composition modulates transcription of a geneassociated with an anchor sequence-mediated conjunction, e.g., activatesor represses transcription, e.g., induces epigenetic changes tochromatin.

Anchor Sequence-Mediated Conjunction

In some embodiments, an anchor sequence-mediated conjunction includesone or more anchor sequences, one or more genes, and one or moretranscriptional control sequences, such as an enhancing or silencingsequence. In some embodiments, the transcriptional control sequences iswithin, partially within, or outside the anchor sequence-mediatedconjunction.

In one embodiment, the anchor sequence-mediated conjunction comprises aloop, such as an intra-chromosomal loop. In certain embodiments, theanchor sequence-mediated conjunction has a plurality of loops. One ormore loops may include a first anchor sequence, a nucleic acid sequence,a transcriptional control sequence, and a second anchor sequence. Inanother embodiment, at least one loop includes, in order, a first anchorsequence, a transcriptional control sequence, and a second anchorsequence; or a first anchor sequence, a nucleic acid sequence, and asecond anchor sequence. In yet another embodiment, either one or both ofthe nucleic acid sequences and the transcriptional control sequence islocated within or outside the loop. In still another embodiment, one ormore of the loops comprises a transcriptional control sequence.

In some embodiments, the anchor sequence-mediated conjunction includes aTATA box, a CAAT box, a GC box, or a CAP site.

In some embodiments, the anchor sequence-mediated conjunction comprisesa plurality of loops, and where the anchor sequence-mediated conjunctioncomprises at least one of an anchor sequence, a nucleic acid sequence,and a transcriptional control sequence in one or more of the loops.

In one aspect, the composition described herein may comprise acomposition for introducing a targeted alteration to an anchorsequence-mediated conjunction to modulate transcription of a nucleicacid sequence with a targeting moiety that binds the anchor sequence. Insome embodiments, the anchor sequence-mediated conjunction is altered bytargeting one or more nucleotides within the anchor sequence-mediatedconjunction for substitution, addition or deletion.

In some embodiments, transcription is activated by inclusion of anactivating loop or exclusion of a repressive loop. In one suchembodiment, the anchor sequence-mediated conjunction comprises atranscriptional control sequence that increases transcription of thenucleic acid sequence. In another such embodiment, the anchorsequence-mediated conjunction excludes a transcriptional controlsequence that decreases transcription of the nucleic acid sequence.

In some embodiments, transcription is repressed by inclusion of arepressive loop or exclusion of an activating loop. In one suchembodiment, the anchor sequence-mediated conjunction includes atranscriptional control sequence that decreases transcription of thenucleic acid sequence. In another such embodiment, the anchorsequence-mediated conjunction excludes a transcriptional controlsequence that increases transcription of the nucleic acid sequence.

Anchor Sequence Each anchor sequence-mediated conjunction comprises oneor more anchor sequences, e.g., a plurality. Anchor sequences can bemanipulated or altered to disrupt naturally occurring loops or form newloops (e.g., to form exogenous loops or to form non-naturally occurringloops with exogenous or altered anchor sequences, see FIGS. 3, 4, and5). Such alterations modulate gene expression by changing the2-dimensional structure of DNA, e.g., by thereby modulating the abilityof a target gene to interact with gene regulation and control factors(e.g., enhancing and silencing/repressive sequences). In someembodiments, the chromatin structure is modified by substituting, addingor deleting one or more nucleotides within an anchor sequence of theanchor sequence-mediated conjunction.

The anchor sequences may be non-contiguous with one another. Inembodiments with non-contiguous anchor sequences, the first anchorsequence may be separated from the second anchor sequence by about 500bp to about 500 Mb, about 750 bp to about 200 Mb, about 1 kb to about100 Mb, about 25 kb to about 50 Mb, about 50 kb to about 1 Mb, about 100kb to about 750 kb, about 150 kb to about 500 kb, or about 175 kb toabout 500 kb. In some embodiments, the first anchor sequence isseparated from the second anchor sequence by about 500 bp, 600 bp, 700bp, 800 bp, 900 bp, 1 kb, 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35kb, 40 kb, 45 kb, 50 kb, 55 kb, 60 kb, 65 kb, 70 kb, 75 kb, 80 kb, 85kb, 90 kb, 95 kb, 100 kb, 125 kb, 150 kb, 175 kb, 200 kb, 225 kb, 250kb, 275 kb, 300 kb, 350 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900kb, 1 Mb, 2 Mb, 3 Mb, 4 Mb, 5 Mb, 6 Mb, 7 Mb, 8 Mb, 9 Mb, 10 Mb, 15 Mb,20 Mb, 25 Mb, 50 Mb, 75 Mb, 100 Mb, 200 Mb, 300 Mb, 400 Mb, 500 Mb, orany size therebetween.

In one embodiment, the anchor sequence comprises a common nucleotidesequence, e.g., a CTCF-binding motif:N(T/C/G)N(G/A/T)CC(A/T/G)(C/G)(C/T/A)AG(G/A)(G/T)GG(C/A/T)(G/A)(C/G)(C/T/A)(G/A/C)(SEQ ID NO:1), where N is any nucleotide. A CTCF-binding motif may alsobe in the opposite orientation, e.g.,(G/A/C)(C/T/A)(C/G)(G/A)(C/A/T)GG(G/T)(G/A)GA(C/T/A)(C/G)(A/T/G)CC(G/A/T)N(T/C/G)N(SEQ ID NO:2). In one embodiment, the anchor sequence comprises SEQ IDNO:1 or SEQ ID NO:2 or a sequence at least 75%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% identical to either SEQ ID NO:1 or SEQ ID NO:2.

In some embodiments, the anchor sequence-mediated conjunction comprisesat least a first anchor sequence and a second anchor sequence. The firstanchor sequence and second anchor sequence may each comprise a commonnucleotide sequence, e.g., each comprises a CTCF binding motif. In someembodiments, the first anchor sequence and second anchor sequencecomprise different sequences, e.g., the first anchor sequence comprisesa CTCF binding motif and the second anchor sequence comprises an anchorsequence other than a CTCF binding motif. In some embodiments, eachanchor sequence comprises a common nucleotide sequence and one or moreflanking nucleotides on one or both sides of the common nucleotidesequence.

Two CTCF-binding motifs (e.g., contiguous or non-contiguous CTCF bindingmotifs) that can form a conjunction may be present in the genome in anyorientation, e.g., in the same orientation (tandem) either 5′→3′ (lefttandem, e.g., the two CTCF-binding motifs that comprise SEQ ID NO:1) or3′→5′ (right tandem, e.g., the two CTCF-binding motifs comprise SEQ IDNO:2), or convergent orientation, where one CTCF-binding motif comprisesSEQ ID NO:1 and the other comprises SEQ ID NO:2. CTCFBSDB 2.0: DatabaseFor CTCF binding motifs And Genome Organization(http://insulatordb.uthsc.edu/) can be used to identify CTCF bindingmotifs associated with a target gene.

In some embodiments, the anchor sequence comprises a CTCF binding motifassociated with a target disease gene.

In some embodiments, chromatin structure is modified by substituting,adding or deleting one or more nucleotides within at least one anchorsequence, e.g., a conjunction nucleating molecule binding site. One ormore nucleotides may be specifically targeted, e.g., a targetedalteration, for substitution, addition or deletion within the anchorsequence, e.g., a conjunction nucleating molecule binding site.

In some embodiments, the anchor sequence-mediated conjunction is alteredby changing an orientation of at least one common nucleotide sequence,e.g., a conjunction nucleating molecule binding site.

In some embodiments, the anchor sequence comprises a conjunctionnucleating molecule binding site, e.g., CTCF binding motif, and thetargeting moiety introduces an alteration in at least one conjunctionnucleating molecule binding site, e.g. altering binding affinity for theconjunction nucleating molecule.

In some embodiments, the anchor sequence-mediated conjunction is alteredby introducing an exogenous anchor sequence. Addition of a non-naturallyoccurring or exogenous anchor sequence to form or disrupt a naturallyoccurring anchor sequence-mediated conjunction, e.g., by inducing anon-naturally occurring loop to form that alters transcription of thenucleic acid sequence.

Types of Anchor Sequence-Mediated Conjunctions

In some embodiments, the anchor sequence-mediated conjunction comprisesone or more, e.g., 2, 3, 4, 5, or more, genes.

In some embodiments, the disclosure includes a method of modulatingexpression of a target gene in an anchor sequence-mediated conjunctioncomprising targeting a sequence outside of or that is not part of the orcomprised within the target gene or associated transcriptional controlsequences that influence transcription of the gene, such as targeting ananchor sequence, thereby modulating the gene's expression.

In some embodiments, the disclosure includes a method of modulatingtranscription of a target gene comprising targeting a sequencenon-contiguous with the target gene or associated transcriptionalcontrol sequences that influence transcription of the target gene, suchas targeting an anchor sequence.

In some embodiments, the anchor sequence-mediated conjunction isassociated with one or more, e.g., 2, 3, 4, 5, or more, transcriptionalcontrol sequences. In some embodiments, the target gene isnon-contiguous with one or more of the transcriptional controlsequences. In some embodiments where the gene is non-contiguous with thetranscriptional control sequence, the gene may be separated from one ormore transcriptional control sequences by about 100 bp to about 500 Mb,about 500 bp to about 200 Mb, about 1 kb to about 100 Mb, about 25 kb toabout 50 Mb, about 50 kb to about 1 Mb, about 100 kb to about 750 kb,about 150 kb to about 500 kb, or about 175 kb to about 500 kb. In someembodiments, the gene is separated from the transcriptional controlsequence by about 100 bp, 300 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900bp, 1 kb, 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb,50 kb, 55 kb, 60 kb, 65 kb, 70 kb, 75 kb, 80 kb, 85 kb, 90 kb, 95 kb,100 kb, 125 kb, 150 kb, 175 kb, 200 kb, 225 kb, 250 kb, 275 kb, 300 kb,350 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1 Mb, 2 Mb, 3Mb, 4 Mb, 5 Mb, 6 Mb, 7 Mb, 8 Mb, 9 Mb, 10 Mb, 15 Mb, 20 Mb, 25 Mb, 50Mb, 75 Mb, 100 Mb, 200 Mb, 300 Mb, 400 Mb, 500 Mb, or any sizetherebetween.

In some embodiments, the type of anchor sequence-mediated conjunctionmay help to determine how to modulate gene expression, e.g., choice oftargeting moiety, by altering the anchor sequence-mediated conjunction.For example, some types of anchor sequence-mediated conjunctionscomprise one or more transcription control sequences within the anchorsequence-mediated conjunction. Disruption of such an anchorsequence-mediated conjunction by disrupting the formation of the anchorsequence-mediated conjunction, e.g., altering one or more anchorsequences, is likely to decrease transcription of a target gene withinthe anchor sequence-mediated conjunction.

Type 1

In some embodiments, expression of the target gene is regulated,modulated, or influenced by one or more transcriptional controlsequences associated with the anchor sequence-mediated conjunction. Insome embodiments, the anchor sequence-mediated conjunction comprises oneor more associated genes and one or more transcriptional controlsequences. For example, the target gene and one or more transcriptionalcontrol sequences are located within, at least partially, an anchorsequence-mediated conjunction, e.g., a Type 1 anchor sequence-mediatedconjunction, see FIG. 6. The anchor sequence-mediated conjunctiondepicted in FIG. 6 may also be referred to as a “Type 1, EP subtype.”

In some embodiments, the target gene has a defined state of expression,e.g., in its native state, e.g., in a diseased state. For example, thetarget gene may have a high level of expression. By disrupting theanchor sequence-mediated conjunction, expression of the target gene maybe decreased, e.g., decreased transcription due to conformationalchanges of the DNA previously open to transcription within the anchorsequence-mediated conjunction, e.g., decreased transcription due toconformational changes of the DNA creating additional distance betweenthe target gene and the enhancing sequences. In one embodiment, both thegene associated and one or more transcriptional control sequences, e.g.,enhancing sequences, reside inside the anchor sequence-mediatedconjunction. Disruption of the anchor sequence-mediated conjunctiondecreases expression of the gene. In one embodiment, the gene associatedwith the anchor sequence-mediated conjunction is accessible to one ormore transcriptional control sequences that reside inside, at leastpartially, the anchor sequence-mediated conjunction. Disruption of theanchor sequence-mediated conjunction decreases expression of the gene.

For example, a Type 1 anchor sequence-mediated conjunction comprises agene encoding MYC and disruption of the Type 1 anchor sequence-mediatedconjunction decreases expression of the gene and MYC protein levels. Inanother example, a Type 1 anchor sequence-mediated conjunction comprisesa gene encoding Foxj3 and disruption of the Type 1 anchorsequence-mediated conjunction decreases expression of the gene and Foxj3protein levels.

Type 2

In some embodiments, expression of the target gene is regulated,modulated, or influenced by one or more transcriptional controlsequences associated with, but inaccessible due to the anchorsequence-mediated conjunction. For example, the anchor sequence-mediatedconjunction associated with a gene disrupts the ability of one or moretranscriptional control sequences to regulate, modulate, or influenceexpression of the gene. The transcriptional control sequences may beseparated from the gene, e.g., reside on the opposite side, at leastpartially, e.g., inside or outside, of the anchor sequence-mediatedconjunction as the gene, e.g., the gene is inaccessible to thetranscriptional control sequences due to proximity of the anchorsequence-mediated conjunction. In some embodiments, one or moreenhancing sequences are separated from the gene by the anchorsequence-mediated conjunction, e.g., a Type 2 anchor sequence-mediatedconjunction, see FIG. 6.

In some embodiments, a Type 2 the gene is enclosed within the anchorsequence-mediated conjuction, while the transcriptional control sequence(e.g., enhancing sequence) is not enclosed within the anchorsequence-mediated conjuction. This subtype of Type 2 may be referred toas “Type 2, subtype 1.”

In some embodiments, a Type 2 the the transcriptional control sequence(e.g., enhancing sequence) is enclosed within the anchorsequence-mediated conjuction, while the gene is not enclosed within theanchor sequence-mediated conjuction. This subtype of Type 2 may bereferred to as “Type 2, subtype 2.”

In some embodiments, the gene is inaccessible to one or moretranscriptional control sequences due to the anchor sequence-mediatedconjunction, and disruption of the anchor sequence-mediated conjunctionallows the transcriptional control sequence to regulate, modulate, orinfluence expression of the gene. In one embodiment, the gene is insideand outside the anchor sequence-mediated conjunction and inaccessible tothe one or more transcriptional control sequences. Disruption of theanchor sequence-mediated conjunction increases access of thetranscriptional control sequences to regulate, modulate, or influenceexpression of the gene, e.g., the transcriptional control sequencesincrease expression of the gene. In one embodiment, the gene is insidethe anchor sequence-mediated conjunction and inaccessible to the one ormore transcriptional control sequences residing outside, at leastpartially, the anchor sequence-mediated conjunction. Disruption of theanchor sequence-mediated conjunction increases expression of the gene.In one embodiment, the gene is outside, at least partially, the anchorsequence-mediated conjunction and inaccessible to the one or moretranscriptional control sequences residing inside the anchorsequence-mediated conjunction. Disruption of the anchorsequence-mediated conjunction increases expression of the gene.

In some embodiments, the target gene has a defined state of expression,e.g., in its native state, e.g., in a diseased state. For example, thetarget gene may have a moderate to low level of expression. Bydisrupting the anchor sequence-mediated conjunction, expression of thetarget gene may be modulated, e.g., increased transcription due toconformational changes of the DNA previously closed to transcriptionwithin the anchor sequence-mediated conjunction, e.g., increasedtranscription due to conformational changes of the DNA by bringing theenhancing sequences into closer association with the target gene.

For example, a Type 2 anchor sequence-mediated conjunction comprises agene encoding SCN1a and disruption of the Type 2 anchorsequence-mediated conjunction increases expression of the gene and SCN1aprotein levels. In another example, a Type 2 anchor sequence-mediatedconjunction comprises a gene encoding Serpin1a and disruption of theType 2 anchor sequence-mediated conjunction increases expression of thegene and Serpin1a protein levels. In another example, IL-10 mediatedtolerizing responses may be elicited by altering the anchorsequence-mediated conjunction associated with the IL-10 gene, e.g.,expression of IL-10 may be increased to improve the autoimmunecondition. In another example, IL-6 expression may be increased byaltering its associated anchor sequence-mediated conjunction to bringone or more enhancing sequences into closer proximity to the IL-6 gene.

Type 3

In some embodiments, expression of the target gene is regulated,modulated, or influenced by one or more transcriptional controlsequences associated with the anchor sequence-mediated conjunction, butnot necessarily located on the same side of the anchor sequence-mediatedconjunction as each other. For example, the anchor sequence-mediatedconjunction is associated with one or more genes and one or moretranscriptional control sequences reside inside and outside, at leastpartially, the anchor sequence-mediated conjunction. In someembodiments, one or more enhancing sequences reside inside the anchorsequence-mediated conjunction and one or more repressive signals, e.g.,silencing sequences, reside outside the anchor sequence-mediatedconjunction, e.g., a Type 3 anchor sequence-mediated conjunction, seeFIG. 6.

In some embodiments, the gene is inaccessible to one or moretranscriptional control sequences due to the anchor sequence-mediatedconjunction, and disruption of the anchor sequence-mediated conjunctionallows the transcriptional control sequence to regulate, modulate, orinfluence expression of the gene. In one embodiment, the gene is insidethe anchor sequence-mediated conjunction and inaccessible to the one ormore transcriptional control sequences, e.g., silencing/repressivesequences, residing outside the anchor sequence-mediated conjunction.Disruption of the anchor sequence-mediated conjunction decreasesexpression of the gene. In one embodiment, the gene is inside andoutside the anchor sequence-mediated conjunction and inaccessible to theone or more transcriptional control sequences, e.g.,silencing/repressive sequences, anchor sequence-mediated conjunctionresiding outside the anchor sequence-mediated conjunction. Disruption ofthe anchor sequence-mediated conjunction decreases expression of thegene. In one embodiment, the gene is outside the anchorsequence-mediated conjunction and inaccessible to the one or moretranscriptional control sequences, e.g., silencing/repressive sequences,inside the anchor sequence-mediated conjunction. Disruption of theanchor sequence-mediated conjunction decreases expression of the gene.

In some embodiments, the target gene has a defined state of expression,e.g., in its native state, e.g., in a diseased state. For example, thetarget gene may have a high level of expression in its native state. Bydisrupting the anchor sequence-mediated conjunction, expression of thetarget gene may be modulated, e.g., decreased transcription due toconformational changes of the DNA creating additional distance betweenthe target gene and the enhancing sequences, e.g., decreasedtranscription due to conformational changes of the DNA previously opento transcription within the anchor sequence-mediated conjunction, e.g.,decreased transcription due to conformational changes of the DNAbringing the silencing sequences into closer association with the targetgene, e.g., decreased transcription due to conformational changes of theDNA creating additional distance between the target gene and theenhancing sequences.

Type 4

In some embodiments, expression of the target gene is regulated,modulated, or influenced by one or more transcriptional controlsequences associated with the anchor sequence-mediated conjunction, butnot necessarily located within the anchor sequence-mediated conjunction.For example, the anchor sequence-mediated conjunction is associated withone or more genes and one or more transcriptional control sequencesreside inside and outside, at least partially, the anchorsequence-mediated conjunction, e.g., a Type 4 anchor sequence-mediatedconjunction, see FIG. 6.

In some embodiments, the gene is inaccessible to one or moretranscriptional control sequences due to the anchor sequence-mediatedconjunction, and disruption of the anchor sequence-mediated conjunctionallows the transcriptional control sequence to regulate, modulate, orinfluence expression of the gene. In one embodiment, the gene is insidethe anchor sequence-mediated conjunction and inaccessible to the one ormore transcriptional control sequences residing outside the anchorsequence-mediated conjunction. Disruption of the anchorsequence-mediated conjunction increases expression of the gene. In oneembodiment, the gene is inside and outside the anchor sequence-mediatedconjunction and inaccessible to the one or more transcriptional controlsequences, e.g., enhancing sequences, anchor sequence-mediatedconjunction residing outside the anchor sequence-mediated conjunction.Disruption of the anchor sequence-mediated conjunction increasesexpression of the gene. In one embodiment, the gene is outside theanchor sequence-mediated conjunction and inaccessible to the one or moretranscriptional control sequences, e.g., enhancing sequences, inside theanchor sequence-mediated conjunction. Disruption of the anchorsequence-mediated conjunction increases expression of the gene.

In some embodiments, the target gene has a defined state of expression,e.g., in its native state, e.g., in a diseased state. For example, thetarget gene may have a high level of expression in its native state. Bydisrupting the anchor sequence-mediated conjunction, expression of thetarget gene may be modulated, e.g., increased transcription due toconformational changes opening the DNA to transcription within theanchor sequence-mediated conjunction, e.g., increased transcription dueto conformational changes of the DNA by bringing one or more enhancingsequences into association with the target gene.

Targeting Moieties

In some embodiments, a composition, agent, fusion molecule, or othermolecule as described herein comprises one or more the targetingmoieties described herein. The targeting moiety may target an anchorsequence-mediated conjunction for alteration of at least one of thefollowing: at least one exogenous anchor sequence; an alteration in atleast one conjunction nucleating molecule binding site, such as byaltering binding affinity for the conjunction nucleating molecule; achange in an orientation of at least one common nucleotide sequence,such as a CTCF binding motif; and a substitution, addition or deletionin at least one anchor sequence, such as a CTCF binding motif.

Those skilled in the art reading the below examples of particular kindsof targeting moieties will understand that, in some embodiments, atargeting moiety is site-specific. That is, in some embodiments, atargeting moiety binds specifically to one or more target anchorsequences (e.g., within a cell) and not to non-targeted anchor sequences(e.g., within the same cell).

The targeting moiety may modulate a specific function, modulate aspecific molecule (e.g., enzyme, protein or nucleic acid), andspecifically bind for localization. The targeting function may act on aspecific molecule, e.g. a molecular target. For example, a targetedtherapeutic may interact with a specific molecule to increase, decreaseor otherwise modulate its function.

In some embodiments, the targeting moiety binds an anchor sequence(e.g., a DNA sequence). In various parts of the present disclosure, theterm “DNA binding moiety” may be used to refer to a targeting moiety.

In some embodiments, a composition, agent, fusion molecule, or othermolecule as described herein comprises a targeting moiety (e.g., gRNA,antisense, oligonucleotides, peptide oligonucleotide conjugates) thatbinds the anchor sequence, and is operably linked to an effector moietythat modulates the formation of a conjunction mediated by the anchorsequence. The targeting moiety may bind an anchor sequence of an anchorsequence-mediated conjunction and alter formation of the anchorsequence-mediated conjunction (e.g., alters affinity of the anchorsequence to a conjunction nucleating molecule, e.g., at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or more). The targeting moiety may be any one of the smallmolecules, peptides, nucleic acids, nanoparticles, aptamers, andpharmacoagents with poor pharmacokinetics described herein.

The targeting moiety may target one or more nucleotides, such as througha gene editing system, of a sequence, e.g., an anchor sequence, e.g., acommon nucleotide sequence within an anchor sequence, within the anchorsequence-mediated conjunction for substitution, addition or deletion. Insome embodiments, the targeting moiety binds an anchor sequence-mediatedconjunction, e.g., the anchor sequence in the anchor sequence-mediatedconjunction, and alters a topology of the anchor sequence-mediatedconjunction.

In some embodiments, the targeting moiety targets one or morenucleotides, e.g., such as through CRISPR, TALEN, dCas9, oligonucleotidepairing, recombination, transposon, etc., of an anchor sequence withinthe anchor sequence-mediated conjunction for substitution, addition ordeletion. In some embodiments, the targeting moiety targets one or moreDNA methylation sites within the anchor sequence-mediated conjunction.

The targeting moiety may alter one or more nucleotides, such as througha gene editing system, of a sequence, e.g., an anchor sequence, e.g., acommon nucleotide sequence within an anchor sequence, within the anchorsequence-mediated conjunction by substitution, addition or deletion.

In some embodiments, the targeting moiety introduces a targetedalteration into the anchor sequence-mediated conjunction to modulatetranscription, in a human cell, of a gene in the anchorsequence-mediated conjunction. The targeted alteration may include asubstitution, addition or deletion of one or more nucleotides, e.g., ofan anchor sequence within the anchor sequence-mediated conjunction. Thetargeting moiety may bind an anchor sequence of the anchorsequence-mediated conjunction and the targeting moiety introduce atargeted alteration into the anchor sequence to modulate transcription,in a human cell, of a gene in the anchor sequence-mediated conjunction.In some embodiments, the targeted alteration alters at least one of abinding site for a conjunction nucleating molecule, e.g. alteringbinding affinity for an anchor sequence within the anchorsequence-mediated conjunction, an alternative splicing site, and abinding site for a non-translated RNA.

In some embodiments, the targeting moiety edits an anchorsequence-mediated conjunction at least one of the following: at leastone exogenous anchor sequence; an alteration in at least one conjunctionnucleating molecule binding site, such as by altering binding affinityfor the conjunction nucleating molecule; a change in an orientation ofat least one common nucleotide sequence, such as a CTCF binding motif;and a substitution, addition or deletion in at least one anchorsequence, such as a CTCF binding motif.

In some embodiments, the targeting moiety is a nucleic acid sequence, aprotein, protein fusion, or a membrane translocating polypeptide. Insome embodiments, the targeting moiety is selected from an exogenousconjunction nucleating molecule, a nucleic acid encoding the conjunctionnucleating molecule, or a fusion of a sequence targeting polypeptide anda conjunction nucleating molecule.

As described in greater detail herein, in some embodiments, a targetingmoiety as described herein can be or comprise a polymer or polymericmoiety, e.g., a polymer of nucleotides (such as an oligonucleotide), apeptide nucleic acid, a peptide-nucleic acid mixmer, a peptide orpolypeptide, a polyamide, a carbohydrate, etc.

Nucleic Acid Sequences

In some embodiments, the targeting moiety comprises a nucleic acidsequence. In some embodiments, the nucleic acid sequence encodes a geneor an expression product.

As will be readily understand by those skilled in the art reading thepresent specification, a targeting moiety can comprise a nucleic acidsequence that does not encode a gene or an expression product. Forexample, in some embodiments, a targeting moiety comprises anoligonucleotide that hybridizes to a target anchor sequence. Forexample, in some embodiments, the sequence of the oligonucleotidecomprises a complement of the target anchor sequence, or has a sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, at least97%, at least 98%, at least 99% identical to the complement of thetarget anchor sequence.

The nucleic acid sequence may include, but is not limited to, DNA, RNA,modified oligonucleotides (e.g., chemical modifications, such asmodifications that alter the backbone linkages, sugar molecules, and/ornucleic acid bases), and artificial nucleic acids. In some embodiments,the nucleic acid sequence includes, but is not limited to, genomic DNA,cDNA, peptide nucleic acids (PNA) or peptide oligonucleotide conjugates,locked nucleic acids (LNA), bridged nucleic acids (BNA), polyamides,triplex forming oligonucleotides, modified DNA, antisense DNAoligonucleotides, tRNA, mRNA, rRNA, modified RNA, miRNA, gRNA, and siRNAor other RNA or DNA molecules.

In some embodiments, the nucleic acid sequence has a length from about 2to about 5000 nts, about 10 to about 100 nts, about 50 to about 150 nts,about 100 to about 200 nts, about 150 to about 250 nts, about 200 toabout 300 nts, about 250 to about 350 nts, about 300 to about 500 nts,about 10 to about 1000 nts, about 50 to about 1000 nts, about 100 toabout 1000 nts, about 1000 to about 2000 nts, about 2000 to about 3000nts, about 3000 to about 4000 nts, about 4000 to about 5000 nts, or anyrange therebetween.

In one aspect, the disclosure includes a synthetic nucleic acidcomprising a plurality of anchor sequences, a gene sequence, and atranscriptional control sequence. In some embodiments, the gene sequenceand the transcriptional control sequence are between the plurality ofanchor sequences. In some embodiments, the synthetic nucleic acidcomprises, in order, (a) an anchor sequence, a gene sequence, atranscriptional control sequence, and an anchor sequence or (b) ananchor sequence, a transcriptional control sequence, a gene sequence,and an anchor sequence. In some embodiments, the sequences are separatedby linker sequences. In some embodiments, the anchor sequences arebetween 7-100 nts, 10-100 nts, 10-80 nts, 10-70 nts, 10-60 nts, 10-50nts, 20-80 nts, or any range therebetween. In some embodiments, thenucleic acid is between 3,000-50,000 bp, 3,000-40,000 bp, 3,000-30,000bp, 3,000-20,000 bp, 3,000-15,000 bp, 3,000-12,000 bp, 3,000-10,000 bp,3,000-8,000 bp, 5,000-30,000 bp, 5,000-20,000 bp, 5,000-15,000 bp,5,000-12,000 bp, 5,000-10,000 bp or any range therebetween.

In another aspect, the disclosure includes a vector comprising thenucleic acid described herein.

In another aspect, the disclosure includes a cell or tissue comprisingthe nucleic acid described herein.

In another aspect, the disclosure includes a pharmaceutical compositioncomprising the nucleic acid described herein.

In another aspect, the disclosure includes a method of modulatingexpression of a gene by administering the composition comprising thenucleic acid described herein.

Analogs

The nucleic acid sequence may include nucleosides, e.g., purines orpyrimidines, e.g., adenine, cytosine, guanine, thymine and uracil. Insome embodiments, the nucleic acid sequence includes one or morenucleoside analogs. The nucleoside analog includes, but is not limitedto, a nucleoside analog, such as 5-fluorouracil; 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,4-methylbenzimidazole, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil, dihydrouridine,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,2,6-diaminopurine, 3-nitropyrrole, inosine, thiouridine, queuosine,wyosine, diaminopurine, isoguanine, isocytosine, diaminopyrimidine,2,4-difluorotoluene, isoquinoline, pyrrolo[2,3-β]pyridine, and anyothers that can base pair with a purine or a pyrimidine side chain.

gRNA

In some embodiments, the targeting moiety comprises a nucleic acidsequence, e.g., a guide RNA (gRNA). In some embodiments, the targetingmoiety comprises a guide RNA or nucleic acid encoding the guide RNA. AgRNA short synthetic RNA composed of a “scaffold” sequence necessary forCas9-binding and a user-defined ˜20 nucleotide targeting sequence for agenomic target. In practice, guide RNA sequences are generally designedto have a length of between 17-24 nucleotides (e.g., 19, 20, or 21nucleotides) and complementary to the targeted nucleic acid sequence.Custom gRNA generators and algorithms are available commercially for usein the design of effective guide RNAs. Gene editing has also beenachieved using a chimeric “single guide RNA” (“sgRNA”), an engineered(synthetic) single RNA molecule that mimics a naturally occurringcrRNA-tracrRNA complex and contains both a tracrRNA (for binding thenuclease) and at least one crRNA (to guide the nuclease to the sequencetargeted for editing). Chemically modified sgRNAs have also beendemonstrated to be effective in genome editing; see, for example, Hendelet al. (2015) Nature Biotechnol., 985-991.

In some embodiments, the nucleic acid sequence comprises a sequencecomplementary to an anchor sequence. In one embodiment, the anchorsequence comprises a CTCF-binding motif or consensus sequence:N(T/C/G)N(G/A/T)CC(A/T/G)(C/G)(C/T/A)AG(G/A)(G/T)GG(C/A/T)(G/A)(C/G)(C/T/A)(G/A/C)(SEQ ID NO:1), where N is any nucleotide. A CTCF-binding motif orconsensus sequence may also be in the opposite orientation, e.g.,(G/A/C)(C/T/A)(C/G)(G/A)(C/A/T)GG(G/T)(G/A)GA(C/T/A)(C/G)(A/T/G)CC(G/A/T)N(T/C/G)N(SEQ ID NO:2). In some embodiments, the nucleic acid sequence comprisesa sequence complementary to a CTCF-binding motif or consensus sequence.

In some embodiments, the nucleic acid sequence comprises a sequence atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% complementary to an anchor sequence. In someembodiments, the nucleic acid sequence comprises a sequence at least80%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% complementary to a CTCF-binding motif or consensussequence. In some embodiments, the nucleic acid sequence is selectedfrom the group consisting of a gRNA, and a sequence complementary or asequence comprising at least 80%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% complementary sequence toan anchor sequence.

In some embodiments, the epigenetic modifying agent is a gRNA, antisenseDNA, or triplex forming oligonucleotide used as a DNA target and stericpresence in the vicinity of the anchoring sequence. The gRNA recognizesspecific DNA sequences (e.g., an anchor sequence, a CTCF anchorsequence, flanked by sequences that confer sequence specificity). ThegRNA may include additional sequences that interfere with conjunctionnucleating molecule sequence to act as a steric blocker. In someembodiments, the gRNA is combined with one or more peptides, e.g.,S-adenosyl methionine (SAM), that acts as a steric presence to interferewith a conjunction nucleating molecule.

Protein Encoding Nucleic Acids

in some embodiments, a vector, e.g., a viral vector, comprises a nucleicacid encoding a targeting moiety, e.g., a conjunction nucleatingmolecule.

The nucleic acids described herein or the nucleic acids encoding aprotein described herein, e.g., conjunction nucleating molecule orepigenetic modifying agent, may be incorporated into a vector. Vectors,including those derived from retroviruses such as lentivirus, aresuitable tools to achieve long-term gene transfer since they allowlong-term, stable integration of a transgene and its propagation indaughter cells. Examples of vectors include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.The expression vector may be provided to a cell in the form of a viralvector. Viral vector technology is well known in the art, and describedin a variety of virology and molecular biology manuals. Viruses, whichare useful as vectors include, but are not limited to, retroviruses,adenoviruses, adeno-associated viruses, herpes viruses, andlentiviruses. In general, a suitable vector contains an origin ofreplication functional in at least one organism, a promoter sequence,convenient restriction endonuclease sites, and one or more selectablemarkers.

Expression of natural or synthetic nucleic acids is typically achievedby operably linking a nucleic acid encoding the gene of interest to apromoter, and incorporating the construct into an expression vector. Thevectors can be suitable for replication and integration in eukaryotes.Typical cloning vectors contain transcription and translationterminators, initiation sequences, and promoters useful for expressionof the desired nucleic acid sequence.

Additional promoter elements, e.g., enhancing sequences, regulate thefrequency of transcriptional initiation. Typically, these are located inthe region 30-110 bp upstream of the start site, although a number ofpromoters have recently been shown to contain functional elementsdownstream of the start site as well. The spacing between promoterelements frequently is flexible, so that promoter function is preservedwhen elements are inverted or moved relative to one another. In thethymidine kinase (tk) promoter, the spacing between promoter elementscan be increased to 50 bp apart before activity begins to decline.Depending on the promoter, it appears that individual elements canfunction either cooperatively or independently to activatetranscription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter.

Further, the disclosure should not be limited to the use of constitutivepromoters. Inducible promoters are also contemplated as part of thedisclosure. The use of an inducible promoter provides a molecular switchcapable of turning on expression of the polynucleotide sequence which itis operatively linked when such expression is desired, or turning offthe expression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

The expression vector to be introduced can also contain either aselectable marker gene or a reporter gene or both to facilitateidentification and selection of expressing cells from the population ofcells sought to be transfected or infected through viral vectors. Inother aspects, the selectable marker may be carried on a separate pieceof DNA and used in a co-transfection procedure. Both selectable markersand reporter genes may be flanked with appropriate transcriptionalcontrol sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes may be used for identifying potentially transfected cellsand for evaluating the functionality of transcriptional controlsequences. In general, a reporter gene is a gene that is not present inor expressed by the recipient source and that encodes a polypeptidewhose expression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

RNAi

Certain RNA agents can inhibit gene expression through the biologicalprocess of RNA interference (RNAi). RNAi molecules comprise RNA orRNA-like structures typically containing 15-50 base pairs (such as about18-25 base pairs) and having a nucleobase sequence identical(complementary) or nearly identical (substantially complementary) to acoding sequence in an expressed target gene within the cell. RNAimolecules include, but are not limited to: short interfering RNAs(siRNAs), double-strand RNAs (dsRNA), micro RNAs (miRNAs), short hairpinRNAs (shRNA), meroduplexes, and dicer substrates (U.S. Pat. Nos.8,084,599 8,349,809 and 8,513,207). In one embodiment, the disclosureincludes a composition to inhibit expression of a gene encoding apolypeptide described herein, e.g., a conjunction nucleating molecule orepigenetic modifying agent.

RNAi molecules comprise a sequence substantially complementary, or fullycomplementary, to all or a fragment of a target gene. RNAi molecules maycomplement sequences at the boundary between introns and exons toprevent the maturation of newly-generated nuclear RNA transcripts ofspecific genes into mRNA for transcription. RNAi molecules complementaryto specific genes can hybridize with the mRNA for that gene and preventits translation. The antisense molecule can be DNA, RNA, or a derivativeor hybrid thereof. Examples of such derivative molecules include, butare not limited to, peptide nucleic acid (PNA) andphosphorothioate-based molecules such as deoxyribonucleic guanidine(DNG) or ribonucleic guanidine (RNG).

RNAi molecules can be provided to the cell as “ready-to-use” RNAsynthesized in vitro or as an antisense gene transfected into cellswhich will yield RNAi molecules upon transcription. Hybridization withmRNA results in degradation of the hybridized molecule by RNAse H and/orinhibition of the formation of translation complexes. Both result in afailure to produce the product of the original gene.

The length of the RNAi molecule that hybridizes to the transcript ofinterest should be around 10 nucleotides, between about 15 or 30nucleotides, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30 or more nucleotides. The degree of identity of theantisense sequence to the targeted transcript should be at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95.

RNAi molecules may also comprise overhangs, i.e. typically unpaired,overhanging nucleotides which are not directly involved in the doublehelical structure normally formed by the core sequences of the hereindefined pair of sense strand and antisense strand. RNAi molecules maycontain 3′ and/or 5′ overhangs of about 1-5 bases independently on eachof the sense strands and antisense strands. In one embodiment, both thesense strand and the antisense strand contain 3′ and 5′ overhangs. Inone embodiment, one or more of the 3′ overhang nucleotides of one strandbase pairs with one or more 5′ overhang nucleotides of the other strand.In another embodiment, the one or more of the 3′ overhang nucleotides ofone strand base do not pair with the one or more 5′ overhang nucleotidesof the other strand. The sense and antisense strands of an RNAi moleculemay or may not contain the same number of nucleotide bases. Theantisense and sense strands may form a duplex wherein the 5′ end onlyhas a blunt end, the 3′ end only has a blunt end, both the 5′ and 3′ends are blunt ended, or neither the 5′ end nor the 3′ end are bluntended. In another embodiment, one or more of the nucleotides in theoverhang contains a thiophosphate, phosphorothioate, deoxynucleotideinverted (3′ to 3′ linked) nucleotide or is a modified ribonucleotide ordeoxynucleotide.

Small interfering RNA (siRNA) molecules comprise a nucleotide sequencethat is identical to about 15 to about 25 contiguous nucleotides of thetarget mRNA. In some embodiments, the siRNA sequence commences with thedinucleotide AA, comprises a GC-content of about 30-70% (about 30-60%,about 40-60%, or about 45%-55%), and does not have a high percentageidentity to any nucleotide sequence other than the target in the genomeof the mammal in which it is to be introduced, for example as determinedby standard BLAST search.

siRNAs and shRNAs resemble intermediates in the processing pathway ofthe endogenous microRNA (miRNA) genes (Bartel, Cell 116:281-297, 2004).In some embodiments, siRNAs can function as miRNAs and vice versa (Zenget al., Mol Cell 9:1327-1333, 2002; Doench et al., Genes Dev 17:438-442,2003). MicroRNAs, like siRNAs, use RISC to downregulate target genes,but unlike siRNAs, most animal miRNAs do not cleave the mRNA. Instead,miRNAs reduce protein output through translational suppression or polyAremoval and mRNA degradation (Wu et al., Proc Natl Acad Sci USA103:4034-4039, 2006). Known miRNA binding sites are within mRNA 3′ UTRs;miRNAs seem to target sites with near-perfect complementarity tonucleotides 2-8 from the miRNA's 5′ end (Rajewsky, Nat Genet 38Suppl:S8-13, 2006; Lim et al., Nature 433:769-773, 2005). This region isknown as the seed region. Because siRNAs and miRNAs are interchangeable,exogenous siRNAs downregulate mRNAs with seed complementarity to thesiRNA (Birmingham et al., Nat Methods 3:199-204, 2006. Multiple targetsites within a 3′ UTR give stronger downregulation (Doench et al., GenesDev 17:438-442, 2003).

Lists of known miRNA sequences can be found in databases maintained byresearch organizations, such as Wellcome Trust Sanger Institute, PennCenter for Bioinformatics, Memorial Sloan Kettering Cancer Center, andEuropean Molecule Biology Laboratory, among others. Known effectivesiRNA sequences and cognate binding sites are also well represented inthe relevant literature. RNAi molecules are readily designed andproduced by technologies known in the art. In addition, there arecomputational tools that increase the chance of finding effective andspecific sequence motifs (Pei et al. 2006, Reynolds et al. 2004,Khvorova et al. 2003, Schwarz et al. 2003, Ui-Tei et al. 2004, Heale etal. 2005, Chalk et al. 2004, Amarzguioui et al. 2004).

The RNAi molecule modulates expression of RNA encoded by a gene. Becausemultiple genes can share some degree of sequence homology with eachother, in some embodiments, the RNAi molecule can be designed to targeta class of genes with sufficient sequence homology. In some embodiments,the RNAi molecule can contain a sequence that has complementarity tosequences that are shared amongst different gene targets or are uniquefor a specific gene target. In some embodiments, the RNAi molecule canbe designed to target conserved regions of an RNA sequence havinghomology between several genes thereby targeting several genes in a genefamily (e.g., different gene isoforms, splice variants, mutant genes,etc.). In some embodiments, the RNAi molecule can be designed to targeta sequence that is unique to a specific RNA sequence of a single gene.

In some embodiments, the RNAi molecule targets a sequence in aconjunction nucleating molecule, e.g., CTCF, cohesin, USF1, YY1,TATA-box binding protein associated factor 3 (TAF3), ZNF143, or anotherpolypeptide that promotes the formation of an anchor sequence-mediatedconjunction, or an epigenetic modifying agent, e.g., an enzyme involvedin post-translational modifications including, but are not limited to,DNA methylases (e.g., DNMT3a, DNMT3b, DNMTL), DNA demethylation (e.g.,the TET family enzymes catalyze oxidation of 5-methylcytosine to5-hydroxymethylcytosine and higher oxidative derivatives), histonemethyltransferases, histone deacetylase (e.g., HDAC1, HDAC2, HDAC3),sirtuin 1, 2, 3, 4, 5, 6, or 7, lysine-specific histone demethylase 1(LSD1), histone-lysine-N-methyltransferase (Setdb1), euchromatichistone-lysine N-methyltransferase 2 (G9a), histone-lysineN-methyltransferase (SUV39H1), enhancer of zeste homolog 2 (EZH2), virallysine methyltransferase (vSET), histone methyltransferase (SET2),protein-lysine N-methyltransferase (SMYD2), and others. In oneembodiment, the RNAi molecule targets a protein deacetylase, e.g.,sirtuin 1, 2, 3, 4, 5, 6, or 7. In one embodiment, the disclosureincludes a composition comprising an RNAi that targets a conjunctionnucleating molecule, e.g., CTCF.

Peptide or Protein Moiety

In some embodiments, the targeting moiety comprises a peptide or proteinmoiety, e.g., a DNA-binding protein, a CRISPR component protein,conjunction nucleating molecule, a dominant negative conjunctionnucleating molecule, an epigenetic modifying agent, or any combinationthereof.

The peptide or protein moieties may include, but is not limited to, apeptide ligand, antibody fragment, or targeting aptamer that binds areceptor such as an extracellular receptor, neuropeptide, hormonepeptide, peptide drug, toxic peptide, viral or microbial peptide,synthetic peptide, and agonist or antagonist peptide.

Peptide or protein moiety may be linear or branched. The peptide orprotein moiety may have a length from about 5 to about 200 amino acids,about 15 to about 150 amino acids, about 20 to about 125 amino acids,about 25 to about 100 amino acids, or any range therebetween.

Exemplary peptide or protein moiety used in the methods and compositionsdescribed herein include, but are not limited to, ubiquitin, bicyclicpeptides as ubiquitin ligase inhibitors, transcription factors, DNA andprotein modification enzymes such as topoisomerases, topoisomeraseinhibitors such as topotecan, DNA methyltransferases such as the DNMTfamily (e.g., DNMT3a, DNMT3b, DNMTL), protein methyltransferases (e.g.,viral lysine methyltransferase (vSET), protein-lysineN-methyltransferase (SMYD2), deaminases (e.g., APOBEC, UG1), histonemethyltransferases such as enhancer of zeste homolog 2 (EZH2), PRMT1,histone-lysine-N-methyltransferase (Setdb1), histone methyltransferase(SET2), euchromatic histone-lysine N-methyltransferase 2 (G9a),histone-lysine N-methyltransferase (SUV39H1), and G9a), histonedeacetylase (e.g., HDAC1, HDAC2, HDAC3), enzymes with a role in DNAdemethylation (e.g., the TET family enzymes catalyze oxidation of5-methylcytosine to 5-hydroxymethylcytosine and higher oxidativederivatives), protein demethylases such as KDM1A and lysine-specifichistone demethylase 1 (LSD1), helicases such as DHX9,acetyltransferases, deacetylases (e.g., sirtuin 1, 2, 3, 4, 5, 6, or 7),kinases, phosphatases, DNA-intercalating agents such as ethidiumbromide, sybr green, and proflavine, efflux pump inhibitors such aspeptidomimetics like phenylalanine arginyl β-naphthylamide or quinolinederivatives, nuclear receptor activators and inhibitors, proteasomeinhibitors, competitive inhibitors for enzymes such as those involved inlysosomal storage diseases, protein synthesis inhibitors, nucleases(e.g., Cpf1, Cas9, zinc finger nuclease), fusions of one or more thereof(e.g., dCas9-DNMT, dCas9-APOBEC, dCas9-UG1), and specific domains fromproteins, such as KRAB domain.

Some examples of peptides include, but are not limited to, fluorescenttags or markers, antigens, antibodies, antibody fragments such as singledomain antibodies, ligands and receptors such as glucagon-like peptide-1(GLP-1), GLP-2 receptor 2, cholecystokinin B (CCKB) and somatostatinreceptor, peptide therapeutics such as those that bind to specific cellsurface receptors such as G protein-coupled receptors (GPCRs) or ionchannels, synthetic or analog peptides from naturally-bioactivepeptides, anti-microbial peptides, pore-forming peptides, tumortargeting or cytotoxic peptides, and degradation or self-destructionpeptides such as an apoptosis-inducing peptide signal or photosensitizerpeptide.

Peptides described herein may also include small antigen-bindingpeptides, e.g., antigen binding antibody or antibody-like fragments,such as single chain antibodies, nobodies (see, e.g., Steeland et al.2016. Nanobodies as therapeutics: big opportunities for smallantibodies. Drug Discov Today: 21(7):1076-113). Such small antigenbinding peptides may bind a cytosolic antigen, a nuclear antigen, anintra-organellar antigen.

In one aspect, the disclosure includes a cell or tissue comprising anyone of the proteins described herein.

In another aspect, the disclosure includes a pharmaceutical compositioncomprising the protein described herein.

In another aspect, the disclosure includes a method of modulatingexpression of a gene by administering the composition comprising theprotein described herein.

DNA-Binding Domains

In some embodiments, the targeting moiety comprises aDNA-binding domainof a protein. DNA-binding proteins have distinct structural motifs thatplay a key role in binding DNA.

The helix-turn-helix motif is a common DNA recognition motif inrepressor proteins. The motif comprises two helices, one of whichrecognizes the DNA (aka recognition helix) and the side chains give thespecificity of binding. They are common in proteins that regulatedevelopmental processes. Sometimes more than one protein competes forthe same sequence or recognizes the same DNA fragment. They may differin their affinity for the same sequence, or DNA conformation,respectively through H-bonds, salt bridges and Van der Waalsinteractions.

DNA-binding proteins with an HhH structural motif may be involved innon-sequence-specific DNA binding that occurs via the formation ofhydrogen bonds between protein backbone nitrogens and DNA phosphategroups.

DNA-binding proteins with the HLH structural motif are transcriptionalregulatory proteins and are principally related to a wide array ofdevelopmental processes. The motif is longer, in terms of residues, thanthe other two motifs. Many of these proteins interact to form homo- andhetero-dimers. The structural motif is composed of two long helixregions, with the N-terminal helix binding to the DNA, while the loopregion allows the protein to dimerize.

In some transcription factors, the dimer binding site with DNA forms aleucine zipper. This motif includes two amphipathic helices, one fromeach subunit, interacting with each other resulting in a left handedcoiled-coil super secondary structure. The leucine zipper is aninterdigitation of regularly spaced leucine residues in one helix withleucines from an adjacent helix. Mostly, the helices involved in leucinezippers exhibit a heptad sequence (abcdefg) with residues a and d beinghydrophobic and all others hydrophilic. Leucine zipper motifs canmediate either homo- or heterodimer formation.

Some eukaryotic transcription factors show a unique motif called aZn-finger, where a Zn⁺⁺ ion is coordinated by 2 Cys and 2 His residues.The transcription factor includes a trimer with the stoichiometry ββ′α.The apparent effect of the Zn⁺⁺ coordination is the stabilization of asmall loop structure instead of hydrophobic core residues. EachZn-finger interacts in a conformationally identical manner withsuccessive triple base pair segments in the major groove of the doublehelix. The protein-DNA interaction is determined by two factors: (i)H-bonding interaction between α-helix and DNA segment, mostly betweenArg residues and Guanine bases. (ii) H-bonding interaction with the DNAphosphate backbone, mostly with Arg and His. An alternative Zn-fingermotif chelates the Zn⁺⁺ with 6 Cys.

DNA-binding proteins also include TATA box binding proteins, firstidentified as a component of the class II initiation factor TFIID. Theyparticipate in transcription by all three nuclear RNA polymerases actingas subunit in each of them. The structure of TBP shows two α/βstructural domains of 89-90 amino acids. The C-terminal or core regionbinds with high affinity to a TATA consensus sequence (TATAa/tAa/t, SEQID NO: xx) recognizing minor groove determinants and promoting DNAbending. TBP resemble a molecular saddle. The binding side is lined withthe central 8 strands of the 10-stranded anti-parallel β-sheet. Theupper surface contains four α-helices and binds to various components ofthe transcription machinery.

DNA provides base specificity in the form of nitrogen bases. TheR-groups of amino acids, with basic residues such as Lysine, Arginine,Histidine, Aspargine and Glutamine can easily interact with adenine ofthe A: T base pair, and guanine of the G: C base pair, where NH2 and X═Ogroups of the base pairs can preferably form hydrogen bonds with aminoacid residues of Glutamine, Aspargine, Arginine and Lysine.

In some embodiments, the DNA-binding protein is a transcription factor.Transcription factors (TFs) may be modular proteins containing aDNA-binding domain that is responsible for the specific recognition ofbase sequences and one or more effector domains that can activate orrepress transcription. TFs interact with chromatin and recruit proteincomplexes that serve as coactivators or corepressors.

Gene Editing Systems

In some embodiments, the targeting moiety (e.g., a site-specifictargeting moiety) comprises one or more components of a gene editingsystem. As can be appreciated by those skilled in the art reading thepresent specification, and as explained further herein, components ofgene editing systems may be used in a variety of contexts including butnot limited to gene editing. For example, such components may be used totarget agents that physically modify, genetically modify, and/orepigenetically modify target anchor sequences,

In some embodiments, the targeting moiety targets one or morenucleotides of the anchor sequence-mediated conjunction forsubstitution, addition and/or deletion. Exemplary gene editing systemsinclude the clustered regulatory interspaced short palindromic repeat(CRISPR) system, zinc finger nucleases (ZFNs), and TranscriptionActivator-Like Effector-based Nucleases (TALEN). ZFNs, TALENs, andCRISPR-based methods are described, e.g., in Gaj et al. TrendsBiotechnol. 31.7(2013):397-405; CRISPR methods of gene editing aredescribed, e.g., in Guan et al., Application of CRISPR-Cas system ingene therapy: Pre-clinical progress in animal model. DNA Repair 2016July 30 [Epub ahead of print]; Zheng et al., Precise gene deletion andreplacement using the CRISPR/Cas9 system in human cells. BioTechniques,Vol. 57, No. 3, September 2014, pp. 115-124.

For example, in some embodiments the site-specific targeting moietycomprises a Cas nuclease (e.g., Cas9) and a site-specific guide RNA, asdescribed further herein. In some embodiments, the Cas nuclease isenzymatically inactive, e.g., a dCas9, as described further herein.

In one embodiment, the methods and compositions described herein can beused with a CRISPR-based gene editing, whereby guide RNA (gRNA) are usedin a clustered regulatory interspaced short palindromic repeat (CRISPR)system for gene editing. CRISPR systems are adaptive defense systemsoriginally discovered in bacteria and archaea. CRISPR systems useRNA-guided nucleases termed CRISPR-associated or “Cas” endonucleases (e.g., Cas9 or Cpf1) to cleave foreign DNA. In a typical CRISPR/Cas system,an endonuclease is directed to a target nucleotide sequence (e. g., asite in the genome that is to be sequence-edited) by sequence-specific,non-coding “guide RNAs” that target single- or double-stranded DNAsequences. Three classes (I-III) of CRISPR systems have been identified.The class II CRISPR systems use a single Cas endonuclease (rather thanmultiple Cas proteins). One class II CRISPR system includes a type IICas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and atrans-activating crRNA (“tracrRNA”). The crRNA contains a “guide RNA”,typically about 20-nucleotide RNA sequence that corresponds to a targetDNA sequence. The crRNA also contains a region that binds to thetracrRNA to form a partially double-stranded structure which is cleavedby RNase III, resulting in a crRNA/tracrRNA hybrid. The crRNA/tracrRNAhybrid then directs the Cas9 endonuclease to recognize and cleave thetarget DNA sequence. The target DNA sequence must generally be adjacentto a “protospacer adjacent motif” (“PAM”) that is specific for a givenCas endonuclease; however, PAM sequences appear throughout a givengenome. CRISPR endonucleases identified from various prokaryotic specieshave unique PAM sequence requirements; examples of PAM sequences include5′-NGG (Streptococcus pyogenes), 5′-NNAGAA (Streptococcus thermophilusCRISPR1), 5′-NGGNG (Streptococcus thermophilus CRISPR3), and 5′-NNNGATT(Neisseria meningiditis). Some endonucleases, e. g., Cas9 endonucleases,are associated with G-rich PAM sites, e. g., 5′-NGG, and performblunt-end cleaving of the target DNA at a location 3 nucleotidesupstream from (5′ from) the PAM site. Another class II CRISPR systemincludes the type V endonuclease Cpf1, which is smaller than Cas9;examples include AsCpf1 (from Acidaminococcus sp.) and LbCpf1 (fromLachnospiraceae sp.). Cpf1-associated CRISPR arrays are processed intomature crRNAs without the requirement of a tracrRNA; in other words aCpf1 system requires only the Cpf1 nuclease and a crRNA to cleave thetarget DNA sequence. Cpf1 endonucleases, are associated with T-rich PAMsites, e. g., 5′-TTN. Cpf1 can also recognize a 5′-CTA PAM motif. Cpf1cleaves the target DNA by introducing an offset or staggereddouble-strand break with a 4- or 5-nucleotide 5′ overhang, for example,cleaving a target DNA with a 5-nucleotide offset or staggered cutlocated 18 nucleotides downstream from (3′ from) from the PAM site onthe coding strand and 23 nucleotides downstream from the PAM site on thecomplimentary strand; the 5-nucleotide overhang that results from suchoffset cleavage allows more precise genome editing by DNA insertion byhomologous recombination than by insertion at blunt-end cleaved DNA.See, e. g., Zetsche et al. (2015) Cell, 163:759-771.

A variety of CRISPR associated (Cas) genes or proteins can be used inthe methods of the disclosure and the choice of Cas protein will dependupon the particular conditions of the method. Specific examples of Casproteins include class II systems including Cas1, Cas2, Cas3, Cas4,Cas5, Cash, Cas7, Cas8, Cas9, Cas10, Cpf1, C2C1, or C2C3. In someembodiments, a Cas protein, e.g., a Cas9 protein, may be from any of avariety of prokaryotic species. In some embodiments a particular Casprotein, e.g., a particular Cas9 protein, is selected to recognize aparticular protospacer-adjacent motif (PAM) sequence. In someembodiments, the targeting moiety includes a sequence targetingpolypeptide, such as an enzyme, e.g., Cas9. In certain embodiments a Casprotein, e.g., a Cas9 protein, may be obtained from a bacteria orarchaea or synthesized using known methods. In certain embodiments, aCas protein may be from a gram positive bacteria or a gram negativebacteria. In certain embodiments, a Cas protein may be from aStreptococcus, (e.g., a S. pyogenes, a S. thermophilus) a Crptococcus, aCorynebacterium, a Haemophilus, a Eubacterium, a Pasteurella, aPrevotella, a Veillonella, or a Marinobacter. In some embodimentsnucleic acids encoding two or more different Cas proteins, or two ormore Cas proteins, may be introduced into a cell, zygote, embryo, oranimal, e.g., to allow for recognition and modification of sitescomprising the same, similar or different PAM motifs. In someembodiments, the Cas protein is modified to deactivate the nuclease,e.g., nuclease-deficient Cas9, and to recruit transcription activatorsor repressors, e.g., the ω-subunit of the E. coli Pol, VP64, theactivation domain of p65, KRAB, or SID4X, to induce epigeneticmodifications, e.g., histone acetyltransferase, histonemethyltransferase and demethylase, DNA methyltransferase and enzyme witha role in DNA demethylation (e.g., the TET family enzymes catalyzeoxidation of 5-methylcytosine to 5-hydroxymethylcytosine and higheroxidative derivatives).

For the purposes of gene editing, CRISPR arrays can be designed tocontain one or multiple guide RNA sequences corresponding to a desiredtarget DNA sequence; see, for example, Cong et al. (2013) Science,339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308. At leastabout 16 or 17 nucleotides of gRNA sequence are required by Cas9 for DNAcleavage to occur; for Cpf1 at least about 16 nucleotides of gRNAsequence is needed to achieve detectable DNA cleavage.

Whereas wild-type Cas9 generates double-strand breaks (DSBs) at specificDNA sequences targeted by a gRNA, a number of CRISPR endonucleaseshaving modified functionalities are available, for example: a “nickase”version of Cas9 generates only a single-strand break; a catalyticallyinactive Cas9 (“dCas9”) does not cut the target DNA but interferes withtranscription by steric hindrance. dCas9 can further be fused with aheterologous effector to repress (CRISPRi) or activate (CRISPRa)expression of a target gene. For example, Cas9 can be fused to atranscriptional silencer (e.g., a KRAB domain) or a transcriptionalactivator (e.g., a dCas9-VP64 fusion). A catalytically inactive Cas9(dCas9) fused to FokI nuclease (“dCas9-FokI”) can be used to generateDSBs at target sequences homologous to two gRNAs. See, e. g., thenumerous CRISPR/Cas9 plasmids disclosed in and publicly available fromthe Addgene repository (Addgene, 75 Sidney St., Suite 550A, Cambridge,Mass. 02139; addgene.org/crispr/). A “double nickase” Cas9 thatintroduces two separate double-strand breaks, each directed by aseparate guide RNA, is described as achieving more accurate genomeediting by Ran et al. (2013) Cell, 154:1380-1389.

CRISPR technology for editing the genes of eukaryotes is disclosed in USPatent Application Publications 2016/0138008A1 and US2015/0344912A1, andin U.S. Pat. Nos. 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233,8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814,8,795,965, and 8,906,616. Cpf1 endonuclease and corresponding guide RNAsand PAM sites are disclosed in US Patent Application Publication2016/0208243 A1.

In some embodiments, the desired genome modification involves homologousrecombination, wherein one or more double-stranded DNA breaks in thetarget nucleotide sequence is generated by the RNA-guided nuclease andguide RNA(s), followed by repair of the break(s) using a homologousrecombination mechanism (“homology-directed repair”). In suchembodiments, a donor template that encodes the desired nucleotidesequence to be inserted or knocked-in at the double-stranded break isprovided to the cell or subject; examples of suitable templates includesingle-stranded DNA templates and double-stranded DNA templates (e. g.,linked to the polypeptide described herein). In general, a donortemplate encoding a nucleotide change over a region of less than about50 nucleotides is provided in the form of single-stranded DNA; largerdonor templates (e. g., more than 100 nucleotides) are often provided asdouble-stranded DNA plasmids. In some embodiments, the donor template isprovided to the cell or subject in a quantity that is sufficient toachieve the desired homology-directed repair but that does not persistin the cell or subject after a given period of time (e. g., after one ormore cell division cycles). In some embodiments, a donor template has acore nucleotide sequence that differs from the target nucleotidesequence (e. g., a homologous endogenous genomic region) by at least 1,at least 5, at least 10, at least 20, at least 30, at least 40, at least50, or more nucleotides. This core sequence is flanked by “homologyarms” or regions of high sequence identity with the targeted nucleotidesequence; in embodiments, the regions of high identity include at least10, at least 50, at least 100, at least 150, at least 200, at least 300,at least 400, at least 500, at least 600, at least 750, or at least 1000nucleotides on each side of the core sequence. In some embodiments wherethe donor template is in the form of a single-stranded DNA, the coresequence is flanked by homology arms including at least 10, at least 20,at least 30, at least 40, at least 50, at least 60, at least 70, atleast 80, or at least 100 nucleotides on each side of the core sequence.In embodiments where the donor template is in the form of adouble-stranded DNA, the core sequence is flanked by homology armsincluding at least 500, at least 600, at least 700, at least 800, atleast 900, or at least 1000 nucleotides on each side of the coresequence. In one embodiment, two separate double-strand breaks areintroduced into the cell or subject's target nucleotide sequence with a“double nickase” Cas9 (see Ran et al. (2013) Cell, 154:1380-1389),followed by delivery of the donor template.

In some embodiments, the composition comprises a polypeptide describedherein linked to a gRNA and a targeted nuclease, e.g., a Cas9, e.g., awild type Cas9, a nickase Cas9 (e.g., Cas9 D10A), a dead Cas9 (dCas9),eSpCas9, Cpf1, C2C1, or C2C3, or a nucleic acid encoding such anuclease. The choice of nuclease and gRNA(s) is determined by whetherthe targeted mutation is a deletion, substitution, or addition ofnucleotides, e.g., a deletion, substitution, or addition of nucleotidesto a targeted sequence. Fusions of a catalytically inactive endonucleasee.g., a dead Cas9 (dCas9, e.g., D10A; H840A) tethered with all or aportion of (e.g., biologically active portion of) an (one or more)effector domain (e.g., epigenome editors including but not restrictedto: DNMT3a, DNMT3L, DNMT3b, KRAB domain, Tet1, p300, VP64 and fusions ofthe aforementioned) create chimeric proteins that can be linked to thepolypeptide to guide the composition to specific DNA sites by one ormore RNA sequences (e.g., DNA recognition elements including, but notrestricted to zinc finger arrays, sgRNA, TAL arrays, peptide nucleicacids described herein) to modulate activity and/or expression of one ormore target nucleic acids sequences (e.g., to methylate or demethylate aDNA sequence).

As used herein, a “biologically active portion of an effector domain” isa portion that maintains the function (e.g. completely, partially,minimally) of an effector domain (e.g., a “minimal” or “core” domain).In some embodiments, fusion of a dCas9 with all or a portion of one ormore effector domains of an epigenetic modifying agent (such as a DNAmethylase or enzyme with a role in DNA demethylation, e.g., DNMT3a,DNMT3b, DNMT3L, a DNMT inhibitor, combinations thereof, TET familyenzymes, protein acetyl transferase or deacetylase, dCas9-DNMT3a/3L,dCas9-DNMT3a/3L/KRAB, dCas9/VP64) creates a chimeric protein that islinked to the polypeptide and useful in the methods described herein.Accordingly, in some embodiments, a nucleic acid encoding adCas9-methylase fusion is linked to the polypeptide and administered toa subject in need thereof in combination with a site-specific gRNA orantisense DNA oligonucleotide that targets the fusion to an anchorsequence (such as a CTCF binding motif), thereby decreasing the affinityor ability of the anchor sequence to bind a nucleating protein. In othersome embodiments, a nucleic acid encoding a dCas9-enzyme fusion islinked to the polypeptide in combination with a site-specific gRNA orantisense DNA oligonucleotide that targets the fusion to a conjunctionanchor sequence (such as a CTCF binding motif) and all are administeredto a subject in need thereof, thereby increasing the affinity or abilityof the anchor sequence to bind a nucleating protein. In someembodiments, all or a portion of one or more methyltransferase, orenzyme associated with demethylation, effector domains are fused with aninactive nuclease, e.g., dCas9, and linked to the polypeptide. ExemplarydCAs9 fusion methods and compositions that are adaptable to the methodsand compositions described herein are known and are described, e.g., inKearns et al., Functional annotation of native enhancers with aCas9-histone demethylase fusion. Nature Methods 12, 401-403 (2015); andMcDonald et al., Reprogrammable CRISPR/Cas9-based system for inducingsite-specific DNA methylation. Biology Open 2016: doi:10.1242/bio.019067.

In other aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, or more methyltransferase, or enzyme with a role in DNAdemethylation, effector domains (all or a biologically active portion)are fused with dCas9 and linked to the polypeptide. The chimericproteins described herein may also comprise a linker as describedherein, e.g., an amino acid linker. In some aspects, a linker comprises2 or more amino acids, e.g., one or more GS sequences. In some aspects,fusion of Cas9 (e.g., dCas9) with two or more effector domains (e.g., ofa DNA me thylase or enzyme with a role in DNA demethylation) comprisesone or more interspersed linkers (e.g., GS linkers) between the domainsand is linked to the polypeptide. In some aspects, dCas9 is fused with aplurality (e.g., 2-5, e.g., 2, 3, 4, 5) effector domains withinterspersed linkers and is linked to the polypeptide.

In some embodiments, a targeting moiety comprises one or more componentsof a CRISPR system described hereinabove.

For example, in some embodiments, a targeting moiety comprises a gRNAthat comprises a targeting domain that hybridizes to a nucleic acidcomprising a target anchor sequence and/or has a sequence that is atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 98%, at least 99% identical to the complement of the nucleic acidcomprising the target anchor sequence. In some embodiments, the gRNA isa site-specific gRNA in that its targeting domain does not hybridize toat least one nucleic acid comprising a non-target anchor sequence.

In some embodiments, the site-specific gRNA comprises a sequence ofstructure I:

X—Y—Z,  (I)

-   -   where X and Z are 5′ and 3′ site specific targeting sequences        for a target CTCF binding motif, respectively, and Y is selected        from:    -   (a) an RNA sequence complementary to the sequence of SEQ ID        NO:1;    -   (b) an RNA sequence at least 75%, 80%, 85%, 90%, 95% identical        to an RNA sequence complementary to the sequence of SEQ ID NO:1;    -   (c) an RNA sequence complementary to the sequence of SEQ ID NO:1        having at least 1, 2, 3, 4, 5, but less than 15, 12 or 10        nucleotide additions, substitutions or deletions.    -   (d) an RNA sequence complementary to the sequence of SEQ ID        NO:2;    -   (e) an RNA sequence at least 75%, 80%, 85%, 90%, 95% identical        to an RNA sequence complementary to the sequence of SEQ ID NO:2;    -   (f) an RNA sequence complementary to the sequence of SEQ ID NO:2        having at least 1, 2, 3, 4, 5, but less than 15, 12 or 10        nucleotide additions, substitutions or deletions.

In some embodiments, X and Z are each between 2-50 nucleotides inlength, e.g., between 2-20, between 2-10, between 2-5 nucleotides inlength.

In some embodiments, a composition or method is described comprising agRNA that specifically targets a CTCF binding motif associated with anoncogene, a tumor suppressor, or a disease associated with a nucleotiderepeat, e.g., CTCFBSDB 2.0: Database For CTCF Binding Motifs And GenomeOrganization.

In some embodiments, provided are pharmaceutical compositions comprisingguide RNAs as described herein.

In some embodiments, the methods described herein include a method ofdelivering one or more CRISPR system component described hereinabove toa subject, e.g., to the nucleus of a cell or tissue of a subject, bylinking such component to a polypeptide described herein.

Conjunction Nucleating Molecules

In some embodiments, the targeting moiety comprises a conjunctionnucleating molecule, a nucleic acid encoding a conjunction nucleatingmolecule, or a combination thereof. In some embodiments, an anchorsequence-mediated conjunction is mediated by a first conjunctionnucleating molecule bound to the first anchor sequence, a secondconjunction nucleating molecule bound to the non-contiguous secondanchor sequence, and an association between the first and secondconjunction nucleating molecules. In some embodiments, a conjunctionnucleating molecule may disrupt, e.g., by competitive binding, thebinding of an endogenous conjunction nucleating molecule to its bindingsite.

The conjunction nucleating molecule may be, e.g., CTCF, cohesin, USF1,YY1, TATA-box binding protein associated factor 3 (TAF3), ZNF143 bindingmotif, or another polypeptide that promotes the formation of an anchorsequence-mediated conjunction. The conjunction nucleating molecule maybe an endogenous polypeptide or other protein, such as a transcriptionfactor, e.g., autoimmune regulator (AIRE), another factor, e.g.,X-inactivation specific transcript (XIST), or an engineered polypeptidethat is engineered to recognize a specific DNA sequence of interest,e.g., having a zinc finger, leucine zipper or bHLH domain for sequencerecognition. The conjunction nucleating molecule may modulate DNAinteractions within or around the anchor sequence-mediated conjunction.For example, the conjunction nucleating molecule can recruit otherfactors to the anchor sequence that alters an anchor sequence-mediatedconjunction formation or disruption.

The conjunction nucleating molecule may also have a dimerization domainfor homo- or heterodimerization. One or more conjunction nucleatingmolecules, e.g., endogenous and engineered, may interact to form theanchor sequence-mediated conjunction. In some embodiments, theconjunction nucleating molecule is engineered to further include astabilization domain, e.g., cohesion interaction domain, to stabilizethe anchor sequence-mediated conjunction. In some embodiments, theconjunction nucleating molecule is engineered to bind a target sequence,e.g., target sequence binding affinity is modulated. In someembodiments, the conjunction nucleating molecule is selected orengineered with a selected binding affinity for an anchor sequencewithin the anchor sequence-mediated conjunction.

Conjunction nucleating molecules and their corresponding anchorsequences may be identified through the use of cells that harborinactivating mutations in CTCF and Chromosome Conformation Capture or3C-based methods, e.g., Hi-C or high-throughput sequencing, to examinetopologically associated domains, e.g., topological interactions betweendistal DNA regions or loci, in the absence of CTCF. Long-range DNAinteractions may also be identified. Additional analyses may includeChIA-PET analysis using a bait, such as Cohesin, YY1 or USF1, ZNF143binding motif, and MS to identify complexes that are associated with thebait.

In some embodiments, one or more conjunction nucleating molecules have abinding affinity for an anchor sequence greater than or less than areference value, e.g., binding affinity for the anchor sequence in theabsence of the alteration.

In some embodiments, the conjunction nucleating molecule is modulated,e.g. a binding affinity for an anchor sequence within the anchorsequence-mediated conjunction, to alter its interaction with the anchorsequence-mediated conjunction.

In some embodiments

Heterologous Moiety

In some embodiments, the composition, agent, and/or fusion moleculedescribed herein may include one or more heterologous moiety. Aheterologous moiety may be an effector (e.g., a drug, small molecule), atag (e.g., fluorophore, light sensitive agent such as KillerRed), or anyof the editing moieties or targeting moieties described herein.

In some embodiments, the heterologous moiety may be linked to a membranetranslocating polypeptide as described herein. In some embodiments, amembrane translocating polypeptide described herein is linked to one ormore heterologous moieties.

In one aspect, the disclosure includes a cell or tissue comprising anyone of the heterologous moieties described herein.

In another aspect, the disclosure includes a pharmaceutical compositioncomprising the heterologous moiety described herein.

In another aspect, the disclosure includes a method of modulatingexpression of a gene by administering the composition comprising theheterologous moiety described herein.

In one aspect, the heterologous moiety is any of the targeting moietiesthat modulate the two-dimensional structure of chromatin (i.e., thatmodulate the structure of chromatin in a way that would alter itstwo-dimensional representation).

In one embodiment, the heterologous moiety is a small molecule (e.g., apeptidomimetic or a small organic molecule with a molecular weight ofless than 2000 daltons), a peptide or polypeptide (e.g., a non ABX^(n)Cpolypeptide, e.g., an antibody or antigen-binding fragment thereof), anucleic acid (e.g., siRNA, mRNA, RNA, DNA, modified DNA or RNA,antisense DNA oligonucleotides, an antisense RNA, a ribozyme, atherapeutic mRNA encoding a protein), a nanoparticle, an aptamer, orpharmacoagent with poor PK/PD.

In some embodiments, the heterologous moiety may cleaved from thepolypeptide (e.g., after administration) by specific proteolysis orenzymatic cleavage (e.g. by TEV protease, Thrombin, Factor Xa orEnteropeptidase).

Effector Moiety

A heterologous moiety may be an effector moiety that possesses effectoractivity. The effector moiety may modulate a biological activity, forexample increasing or decreasing enzymatic activity, gene expression,cell signaling, and cellular or organ function. Effector activities mayalso include binding regulatory proteins to modulate activity of theregulator, such as transcription or translation. Effector activitiesalso may include activator or inhibitor (or “negative effector”)functions as described herein. For example, the heterologous moiety mayinduce enzymatic activity by triggering increased substrate affinity inan enzyme, e.g., fructose 2,6-bisphosphate activates phosphofructokinase1 and increases the rate of glycolysis in response to the insulin. Inanother example, the heterologous moiety may inhibit substrate bindingto a receptor and inhibit its activation, e.g., naltrexone and naloxonebind opioid receptors without activating them and block the receptors'ability to bind opioids. Effector activities may also include modulatingprotein stability/degradation and/or transcript stability/degradation.For example, proteins may be targeted for degradation by the polypeptideco-factor, ubiquitin, onto proteins to mark them for degradation. Inanother example, the heterologous moiety inhibits enzymatic activity byblocking the enzyme's active site, e.g., methotrexate is a structuralanalog of tetrahydrofolate, a coenzyme for the enzyme dihydrofolatereductase that binds to dihydrofolate reductase 1000-fold more tightlythan the natural substrate and inhibits nucleotide base synthesis.

In some embodiments, the composition comprises a targeting moiety (e.g.,gRNA, membrane translocating polypeptide) that binds the anchorsequence, and is operably linked to an effector moiety that modulatesthe formation of a conjunction mediated by the anchor sequence.

In some embodiments, the effector moiety is a chemical, e.g., a chemicalthat modulates a cytosine (C) or an adenine(A) (e.g., Na bisulfite,ammonium bisulfite). In some embodiments, the effector moiety hasenzymatic activity (methyl transferase, demethylase, nuclease (e.g.,Cas9), a deaminase). In some embodiments, the effector moiety stericallyhinders formation of the anchor sequence-mediated conjunction. [e.g.,membrane translocating polypeptide+nanoparticle (def: 1-100 nm)].

The effector moiety with effector activity may be any one of the smallmolecules, peptides, nucleic acids, nanoparticles, aptamers, andpharmacoagents with poor PK/PD described herein.

Negative Effector Moieties

In some embodiments, the effector is an inhibitor or “negativeeffector”. In the context of a negative effector moiety that modulatesformation of an anchor sequence-mediated conjunction, in someembodiments, the negative effector moiety is characterized in thatdimerization of an endogenous nucleating polypeptide is reduced when thenegative effector moiety is present as compared with when it is absent.For example, in some embodiments, the negative effector moiety is orcomprises a variant of the endogenous nucleating polypeptide'sdimerization domain, or a dimerizing portion thereof.

Dominant Negative Conjunction Nucleating Molecules For example, incertain embodiments, an anchor sequence-mediated conjunction is altered(e.g., disrupted) by use of a dominant negative effector, e.g., aprotein that recognizes and binds an anchor sequence, (e.g., a CTCFbinding motif), but with an inactive (e.g., mutated) dimerizationdomain, e.g., a dimerization domain that is unable to form a functionalanchor sequence-mediated conjunction. For example, the Zinc Fingerdomain of CTCF can be altered so that it binds a specific anchorsequence (by adding zinc fingers that recognize flanking nucleic acids),while the homo-dimerization domain is altered to prevent the interactionbetween the engineered CTCF and endogenous forms of CTCF. DNA encodingthe protein can be administered to a subject in need thereof.

In some embodiments, the composition comprises a synthetic conjunctionnucleating molecule with a selected binding affinity for an anchorsequence within a target anchor sequence-mediated conjunction. (thebinding affinity may be at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher or lowerthan the affinity of an endogenous conjunction nucleating molecule thatassociates with the target anchor sequence. The synthetic conjunctionnucleating molecule may have between 30-90%, 30-85%, 30-80%, 30-70%,50-80%, 50-90% amino acid sequence identity to the endogenousconjunction nucleating molecule). The conjunction nucleating moleculemay disrupt, such as through competitive binding, the binding of anendogenous conjunction nucleating molecule to its anchor sequence. Insome more embodiments, the conjunction nucleating molecule is engineeredto bind a novel anchor sequence within the anchor sequence-mediatedconjunction.

In some embodiments, the dominant negative effector has a domain thatrecognizes specific DNA sequences (e.g., an anchor sequence, a CTCFanchor sequence, flanked by sequences that confer sequence specificity),and a second domain that provides a steric presence in the vicinity ofthe anchoring sequence.

The second domain may include a dominant negative conjunction nucleatingmolecule or fragment thereof, a polypeptide that interferes withconjunction nucleating molecule sequence recognition (e.g., the aminoacid backbone of a peptide/nucleic acid or PNA), a nucleic acid sequenceligated to a small molecule that imparts steric interference, or anyother combination of DNA recognition element and a steric blocker.

Epigenetic Modifying Agents

In some embodiments, the heterologous moiety is an epigenetic modifyingagent. Epigenetic modifying agents useful in the methods andcompositions described herein include agents that affect, e.g., DNAmethylation, histone acetylation, and RNA-associated silencing. In someembodiments, the methods described herein involve sequence-specifictargeting of an epigenetic enzyme (e.g., an enzyme that generates orremoves epigenetic marks, e.g., acetylation and/or methylation).Exemplary epigenetic enzymes that can be targeted to an anchor sequenceusing the CRISPR methods described herein include DNA methylases (e.g.,DNMT3a, DNMT3b, DNMTL), DNA demethylation (e.g., the TET family),histone methyltransferases, histone deacetylase (e.g., HDAC1, HDAC2,HDAC3), sirtuin 1, 2, 3, 4, 5, 6, or 7, lysine-specific histonedemethylase 1 (LSD1), histone-lysine-N-methyltransferase (Setdb1),euchromatic histone-lysine N-methyltransferase 2 (G9a), histone-lysineN-methyltransferase (SUV39H1), enhancer of zeste homolog 2 (EZH2), virallysine methyltransferase (vSET), histone methyltransferase (SET2), andprotein-lysine N-methyltransferase (SMYD2). Examples of such epigeneticmodifying agents are described, e.g., in de Groote et al. Nuc. AcidsRes. (2012):1-18.

In some embodiments, an epigenetic modifying agent useful hereincomprises a construct described in Koferle et al. Genome Medicine 7.59(2015):1-3 (e.g., at Table 1), incorporated herein by reference.

Tagging or Monitoring Moiety

A heterologous moiety may be a tag to label or monitor the polypeptidedescribed herein or another heterologous moiety linked to thepolypeptide. The tagging or monitoring moiety may be removable bychemical agents or enzymatic cleavage, such as proteolysis or inteinsplicing. An affinity tag may be useful to purify the tagged polypeptideusing an affinity technique. Some examples include, chitin bindingprotein (CBP), maltose binding protein (MBP), glutathione-S-transferase(GST), and poly(His) tag. A solubilization tag may be useful to aidrecombinant proteins expressed in chaperone-deficient species such as E.coli to assist in the proper folding in proteins and keep them fromprecipitating. Some examples include thioredoxin (TRX) and poly(NANP).The tagging or monitoring moiety may include a light sensitive tag,e.g., fluorescence. Fluorescent tags are useful for visualization. GFPand its variants are some examples commonly used as fluorescent tags.Protein tags may allow specific enzymatic modifications (such asbiotinylation by biotin ligase) or chemical modifications (such asreaction with FlAsH-EDT2 for fluorescence imaging) to occur. Oftentagging or monitoring moiety are combined, in order to connect proteinsto multiple other components. The tagging or monitoring moiety may alsobe removed by specific proteolysis or enzymatic cleavage (e.g. by TEVprotease, Thrombin, Factor Xa or Enteropeptidase).

The tagging or monitoring moiety may be a small molecule, peptide,nucleic acid, nanoparticle, aptamer, or other agent.

Nucleic Acids

A heterologous moiety may be a nucleic acid. A nucleic acid heterologousmoiety may include, but is not limited to, DNA, RNA, and artificialnucleic acids. The nucleic acid may include, but is not limited to,genomic DNA, cDNA, modified DNA, antisense DNA oligonucleotides, tRNA,mRNA, rRNA, modified RNA, miRNA, gRNA, and siRNA or other RNAi molecule.In one embodiment, the nucleic acid is an siRNA to target a geneexpression product. In another embodiment, the nucleic acid includes oneor more nucleoside analogs as described herein.

Nucleic acids have a length from about 2 to about 5000 nts, about 10 toabout 100 nts, about 50 to about 150 nts, about 100 to about 200 nts,about 150 to about 250 nts, about 200 to about 300 nts, about 250 toabout 350 nts, about 300 to about 500 nts, about 10 to about 1000 nts,about 50 to about 1000 nts, about 100 to about 1000 nts, about 1000 toabout 2000 nts, about 2000 to about 3000 nts, about 3000 to about 4000nts, about 4000 to about 5000 nts, or any range therebetween.

Some examples of nucleic acids include, but are not limited to, anucleic acid that hybridizes to an endogenous gene (e.g., gRNA orantisense ssDNA as described herein elsewhere), nucleic acid thathybridizes to an exogenous nucleic acid such as a viral DNA or RNA,nucleic acid that hybridizes to an RNA, nucleic acid that interfereswith gene transcription, nucleic acid that interferes with RNAtranslation, nucleic acid that stabilizes RNA or destabilizes RNA suchas through targeting for degradation, nucleic acid that interferes witha DNA or RNA binding factor through interference of its expression orits function, nucleic acid that is linked to a intracellular protein andmodulates its function, and nucleic acid that is linked to anintracellular protein complex and modulates its function.

The disclosure contemplates the use of RNA therapeutics (e.g., modifiedRNAs) as heterologous moieties useful in the compositions describedherein. For example, a modified mRNA encoding a protein of interest maybe linked to a polypeptide described herein and expressed in vivo in asubject.

In some embodiments, the modified RNA or DNA oligonucleotide linked to apolypeptide described herein, has modified nucleosides or nucleotides.Such modifications are known and are described, e.g., in WO 2012/019168.Additional modifications are described, e.g., in WO2015038892;WO2015038892; WO2015089511; WO2015196130; WO2015196118 andWO2015196128A2.

In some embodiments, the modified RNA or DNA oligonucleotide linked tothe polypeptide described herein has one or more terminal modifications,e.g., a 5′Cap structure and/or a poly-A tail (e.g., of between 100-200nucleotides in length). The 5′ cap structure may be selected from thegroup consisting of CapO, Capl, ARCA, inosine, Nl-methyl-guanosine,2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some cases,the modified RNAs also contains a 5′ UTR comprising at least one Kozaksequence, and a 3′ UTR. Such modifications are known and are described,e.g., in WO2012135805 and WO2013052523. Additional terminalmodifications are described, e.g., in WO2014164253 and WO2016011306.WO2012045075 and WO2014093924.

Chimeric enzymes for synthesizing capped RNA molecules (e.g., modifiedmRNA) which may include at least one chemical modification are describedin WO2014028429.

In some embodiments, a modified mRNA may be cyclized, or concatemerized,to generate a translation competent molecule to assist interactionsbetween poly-A binding proteins and 5′-end binding proteins. Themechanism of cyclization or concatemerization may occur through at least3 different routes: 1) chemical, 2) enzymatic, and 3) ribozymecatalyzed. The newly formed 5′-/3′-linkage may be intramolecular orintermolecular. Such modifications are described, e.g., in WO2013151736.

Methods of making and purifying modified RNAs are known and disclosed inthe art. For example, modified RNAs are made using only in vitrotranscription (IVT) enzymatic synthesis. Methods of making IVTpolynucleotides are known in the art and are described in WO2013151666,WO2013151668, WO2013151663, WO2013151669, WO2013151670, WO2013151664,WO2013151665, WO2013151671, WO2013151672, WO2013151667 andWO2013151736.S Methods of purification include purifying an RNAtranscript comprising a polyA tail by contacting the sample with asurface linked to a plurality of thymidines or derivatives thereofand/or a plurality of uracils or derivatives thereof (polyT/U) underconditions such that the RNA transcript binds to the surface and elutingthe purified RNA transcript from the surface (WO2014152031); using ion(e.g., anion) exchange chromatography that allows for separation oflonger RNAs up to 10,000 nucleotides in length via a scalable method(WO2014144767); and subjecting a modified RMNA sample to DNAse treatment(WO2014152030).

Modified RNAs encoding proteins in the fields of human disease,antibodies, viruses, and a variety of in vivo settings are known and aredisclosed in for example, Table 6 of International Publication Nos.WO2013151666, WO2013151668, WO2013151663, WO2013151669, WO2013151670,WO2013151664, WO2013151665, WO2013151736; Tables 6 and 7 InternationalPublication No. WO2013151672; Tables 6, 178 and 179 of InternationalPublication No. WO2013151671; Tables 6, 185 and 186 of InternationalPublication No WO2013151667. Any of the foregoing may be synthesized asan IVT polynucleotide, chimeric polynucleotide or a circularpolynucleotide and linked to the polypeptide described herein, and eachmay comprise one or more modified nucleotides or terminal modifications.

Peptide Oligonucleotide Conjugates

A heterologous moiety may be a peptide oligonucleotide conjugate.Peptide oligonucleotide conjugates include chimeric molecules comprisinga nucleic acid moiety linked to a peptide moiety (such as apeptide/nucleic acid mixmer). In some embodiments, the peptide moietymay include any peptide or protein moiety described herein. In someembodiments, the nucleic acid moiety may include any nucleic acid oroligonucleotide, e.g., DNA or RNA or modified DNA or RNA, describedherein.

In some embodiments, the peptide oligonucleotide conjugate comprises apeptide antisense oligonucleotide conjugate. In some embodiments, thepeptide oligonucleotide conjugate is a synthetic oligonucleotide with achemically modified backbone. The peptide oligonucleotide conjugate canbind to both DNA and RNA targets in a sequence-specific manner to form aduplex structure. When bound to double-stranded DNA (dsDNA) target, thepeptide oligonucleotide conjugate replaces one DNA strand in the duplexby strand invasion to form a triplex structure and the displaced DNAstrand may exist as a single-stranded D-loop.

In some embodiments, peptide oligonucleotide conjugate may be cell-and/or tissue-specific targeting (which can be conjugated directly tooligos, peptides, and/or proteins, etc.).

In some embodiments, the peptide oligonucleotide conjugate comprises amembrane translocating polypeptide, for example the membranetranslocating polypeptides as described elsewhere herein.

Solid-phase synthesis of several peptide-oligonucleotide conjugates hasbeen described in, for example, Williams, et al., 2010, Curr. Protoc.Nucleic Acid Chem., Chapter Unit 4.41, doi:10.1002/0471142700.nc0441s42. Synthesis and characterization of veryshort peptide-oligonucleotide conjugates and stepwise solid-phasesynthesis of peptide-oligonucleotide conjugates on new solid supportshave been described in, for example, Bongardt, et al., InnovationPerspect. Solid Phase Synth. Comb. Libr., Collect. Pap., Int. Symp.,5th, 1999, 267-270; Antopolsky, et al., Helv. Chim. Acta, 1999, 82,2130-2140.

Nanoparticles

A heterologous moiety may be a nanoparticle. Nanoparticles includeinorganic materials with a size between about 1 and about 1000nanometers, between about 1 and about 500 nanometers in size, betweenabout 1 and about 100 nm, between about 30 nm and about 200 nm, betweenabout 50 nm and about 300 nm, between about 75 nm and about 200 nm,between about 100 nm and about 200 nm, and any range therebetween.Nanoparticle has a composite structure of nanoscale dimensions. In someembodiments, nanoparticles are typically spherical although differentmorphologies are possible depending on the nanoparticle composition. Theportion of the nanoparticle contacting an environment external to thenanoparticle is generally identified as the surface of the nanoparticle.In nanoparticles described herein, the size limitation can be restrictedto two dimensions and so that nanoparticles include composite structurehaving a diameter from about 1 to about 1000 nm, where the specificdiameter depends on the nanoparticle composition and on the intended useof the nanoparticle according to the experimental design. For example,nanoparticles used in therapeutic applications typically have a size ofabout 200 nm or below.

Additional desirable properties of the nanoparticle, such as surfacecharges and steric stabilization, can also vary in view of the specificapplication of interest. Exemplary properties that can be desirable inclinical applications such as cancer treatment are described in Davis etal, Nature 2008 vol. 7, pages 771-782; Duncan, Nature 2006 vol. 6, pages688-701; and Allen, Nature 2002 vol. 2 pages 750-763, each incorporatedherein by reference in its entirety. Additional properties areidentifiable by a skilled person upon reading of the present disclosure.Nanoparticle dimensions and properties can be detected by techniquesknown in the art. Exemplary techniques to detect particles dimensionsinclude but are not limited to dynamic light scattering (DLS) and avariety of microscopies such at transmission electron microscopy (TEM)and atomic force microscopy (AFM). Exemplary techniques to detectparticle morphology include but are not limited to TEM and AFM.Exemplary techniques to detect surface charges of the nanoparticleinclude but are not limited to zeta potential method. Additionaltechniques suitable to detect other chemical properties comprise by ¹H,¹¹B, and ¹³C and ¹⁹F NMR, UV/Vis and infrared/Raman spectroscopies andfluorescence spectroscopy (when nanoparticle is used in combination withfluorescent labels) and additional techniques identifiable by a skilledperson.

Small Molecules

In one embodiment, the targeting moiety is a small molecule that altersone or more DNA methylation sites, e.g., mutates methylated cysteine tothymine, within the anchor sequence-mediated conjunction. For example,bisulfite compounds, e.g., sodium bisulfite, ammonium bisulfite, orother bisulfite salts, may be used to alter one or more DNA methylationsites, e.g., altering the nucleotide sequence from a cysteine to athymine.

A heterologous moiety may be a small molecule. Small molecule moietiesinclude, but are not limited to, small peptides, peptidomimetics (e.g.,peptoids), amino acids, amino acid analogs, synthetic polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic andinorganic compounds (including heterorganic and organomettalliccompounds) generally having a molecular weight less than about 5,000grams per mole, e.g., organic or inorganic compounds having a molecularweight less than about 2,000 grams per mole, e.g., organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, e.g., organic or inorganic compounds having a molecular weightless than about 500 grams per mole, and salts, esters, and otherpharmaceutically acceptable forms of such compounds. Small molecules mayinclude, but are not limited to, a neurotransmitter, a hormone, a drug,a toxin, a viral or microbial particle, a synthetic molecule, andagonists or antagonists.

Examples of suitable small molecules include those described in, “ThePharmacological Basis of Therapeutics,” Goodman and Gilman, McGraw-Hill,New York, N.Y., (1996), Ninth edition, under the sections: Drugs Actingat Synaptic and Neuroeffector Junctional Sites; Drugs Acting on theCentral Nervous System; Autacoids: Drug Therapy of Inflammation; Water,Salts and Ions; Drugs Affecting Renal Function and ElectrolyteMetabolism; Cardiovascular Drugs; Drugs Affecting GastrointestinalFunction; Drugs Affecting Uterine Motility; Chemotherapy of ParasiticInfections; Chemotherapy of Microbial Diseases; Chemotherapy ofNeoplastic Diseases; Drugs Used for Immunosuppression; Drugs Acting onBlood-Forming organs; Hormones and Hormone Antagonists; Vitamins,Dermatology; and Toxicology, all incorporated herein by reference. Someexamples of small molecules include, but are not limited to, prion drugssuch as tacrolimus, ubiquitin ligase or HECT ligase inhibitors such asheclin, histone modifying drugs such as sodium butyrate, enzymaticinhibitors such as 5-aza-cytidine, anthracyclines such as doxorubicin,beta-lactams such as penicillin, anti-bacterials, chemotherapy agents,anti-virals, modulators from other organisms such as VP64, and drugswith insufficient bioavailability such as chemotherapeutics withdeficient pharmacokinetics.

In some embodiments, the small molecule is an epigenetic modifyingagent, for example such as those described in de Groote et al. Nuc.Acids Res. (2012):1-18. Exemplary small molecule epigenetic modifyingagents are described, e.g., in Lu et al. J. Biomolecular Screening17.5(2012):555-71, e.g., at Table 1 or 2, incorporated herein byreference. In some embodiments, an epigenetic modifying agent comprisesvorinostat, romidepsin. In some embodiments, an epigenetic modifyingagent comprises an inhibitor of class I, II, III, and/or IV histonedeacetylase (HDAC). In some embodiments, an epigenetic modifying agentcomprises an activator of SirTI. In some embodiments, an epigeneticmodifying agent comprises Garcinol, Lys-CoA, C646, (+)-JQI, I-BET, BICI,MS120, DZNep, UNC0321, EPZ004777, AZ505, AMI-I, pyrazole amide 7b,benzo[d]imidazole 17b, acylated dapsone derivative (e.e.g, PRMTI),methylstat, 4,4′-dicarboxy-2,2′-bipyridine, SID 85736331, hydroxamateanalog 8, tanylcypromie, bisguanidine and biguanide polyamine analogs,UNC669, Vidaza, decitabine, sodium phenyl butyrate (SDB), lipoic acid(LA), quercetin, valproic acid, hydralazine, bactrim, green tea extract(e.g., epigallocatechin gallate (EGCG)), curcumin, sulforphane and/orallicin/diallyl disulfide. In some embodiments, an epigenetic modifyingagent inhibits DNA methylation, e.g., is an inhibitor of DNAmethyltransferase (e.g., is 5-azacitidine and/or decitabine). In someembodiments, an epigenetic modifying agent modifies histonemodification, e.g., histone acetylation, histone methylation, histonesumoylation, and/or histone phosphorylation. In some embodiments, theepigenetic modifying agent is an inhibitor of a histone deacetylase(e.g., is vorinostat and/or trichostatin A).

In some embodiments, the small molecule is a pharmaceutically activeagent. In one embodiment, the small molecule is an inhibitor of ametabolic activity or component. Useful classes of pharmaceuticallyactive agents include, but are not limited to, antibiotics,anti-inflammatory drugs, angiogenic or vasoactive agents, growth factorsand chemotherapeutic (anti-neoplastic) agents (e.g., tumoursuppressers). One or a combination of molecules from the categories andexamples described herein or from (Orme-Johnson 2007, Methods Cell Biol.2007; 80:813-26) can be used. In one embodiment, the disclosure includesa composition comprising an antibiotic, anti-inflammatory drug,angiogenic or vasoactive agent, growth factor or chemotherapeutic agent.

Oligonucleotide Aptamers

A heterologous moiety may be an oligonucleotide aptamer. Aptamermoieties are oligonucleotide or peptide aptamers. Oligonucleotideaptamers are single-stranded DNA or RNA (ssDNA or ssRNA) molecules thatcan bind to pre-selected targets including proteins and peptides withhigh affinity and specificity.

Oligonucleotide aptamers are nucleic acid species that may be engineeredthrough repeated rounds of in vitro selection or equivalently, SELEX(systematic evolution of ligands by exponential enrichment) to bind tovarious molecular targets such as small molecules, proteins, nucleicacids, and even cells, tissues and organisms. Aptamers providediscriminate molecular recognition, and can be produced by chemicalsynthesis. In addition, aptamers possess desirable storage properties,and elicit little or no immunogenicity in therapeutic applications.

Both DNA and RNA aptamers show robust binding affinities for varioustargets. For example, DNA and RNA aptamers have been selected for tlysozyme, thrombin, human immunodeficiency virus trans-acting responsiveelement (HIV TAR), https://en.widipedia.org/wiki/Aptamer-cite_not-10hemin, interferon γ, vascular endothelial growth factor (VEGF), prostatespecific antigen (PSA), dopamine, and the non-classical oncogene, heatshock factor 1 (HSF1).

Diagnostic techniques for aptamer based plasma protein profilingincludes aptamer plasma proteomics. This technology will enable futuremulti-biomarker protein measurements that can aid diagnostic distinctionof disease versus healthy states.

Peptide Aptamers

A heterologous moiety may be a peptide aptamer. Peptide aptamers haveone (or more) short variable peptide domains, including peptides havinglow molecular weight, 12-14 kDa. Peptide aptamers may be designed tospecifically bind to and interfere with protein-protein interactionsinside cells.

Peptide aptamers are artificial proteins selected or engineered to bindspecific target molecules. These proteins include of one or more peptideloops of variable sequence. They are typically isolated fromcombinatorial libraries and often subsequently improved by directedmutation or rounds of variable region mutagenesis and selection. Invivo, peptide aptamers can bind cellular protein targets and exertbiological effects, including interference with the normal proteininteractions of their targeted molecules with other proteins. Inparticular, a variable peptide aptamer loop attached to a transcriptionfactor binding domain is screened against the target protein attached toa transcription factor activating domain. In vivo binding of the peptideaptamer to its target via this selection strategy is detected asexpression of a downstream yeast marker gene. Such experiments identifyparticular proteins bound by the aptamers, and protein interactions thatthe aptamers disrupt, to cause the phenotype. In addition, peptideaptamers derivatized with appropriate functional moieties can causespecific post-translational modification of their target proteins, orchange the subcellular localization of the targets

Peptide aptamers can also recognize targets in vitro. They have founduse in lieu of antibodies in biosensors and used to detect activeisoforms of proteins from populations containing both inactive andactive protein forms. Derivatives known as tadpoles, in which peptideaptamer “heads” are covalently linked to unique sequence double-strandedDNA “tails”, allow quantification of scarce target molecules in mixturesby PCR (using, for example, the quantitative real-time polymerase chainreaction) of their DNA tails.

Peptide aptamer selection can be made using different systems, but themost used is currently the yeast two-hybrid system. Peptide aptamers canalso be selected from combinatorial peptide libraries constructed byphage display and other surface display technologies such as mRNAdisplay, ribosome display, bacterial display and yeast display. Theseexperimental procedures are also known as biopannings. Among peptidesobtained from biopannings, mimotopes can be considered as a kind ofpeptide aptamers. All the peptides panned from combinatorial peptidelibraries have been stored in a special database with the name MimoDB.

Pharmacoagents

In one embodiment, the heterologous moiety is an agent with anundesirable pharmacokinetic or pharmacodynamics (PK/PD) parameter.Linking the heterologous moiety to the polypeptide may improve at leastone PK/PD parameter, such as targeting, absorption, and transport of theheterologous moiety, or reduce at least one undesirable PK/PD parameter,such as diffusion to off-target sites, and toxic metabolism. Forexample, linking a polypeptide as described herein to an agent with poortargeting/transport, e.g., doxorubicin, beta-lactams such as penicillin,improves its specificity. In another example, linking a polypeptide asdescribed herein to an agent with poor absorption properties, e.g.,insulin, human growth hormone, improves its minimum dosage. In anotherexample, linking a polypeptide as described herein to an agent that hastoxic metabolic properties, e.g., acetaminophen at higher doses,improves its maximum dosage.

Membrane Translocating Polypeptide

In one aspect, the composition comprises a polypeptide described hereinwith properties that allow translocation across a membrane, for example,independent of endosomes, such that the composition is delivered to atarget location within a cell, e.g., within a subject. In someembodiments, the targeting moiety comprises a membrane translocatingpolypeptide.

In one aspect, the disclosure includes a cell or tissue comprising anyone of the membrane translocating polypeptides described herein.

In another aspect, the disclosure includes a pharmaceutical compositioncomprising the membrane translocating polypeptide described herein.

In another aspect, the disclosure includes a method of modulatingexpression of a gene by administering the composition comprising themembrane translocating polypeptide described herein.

In one aspect, the disclosure includes a method altering gene expressionor altering an anchor sequence-mediated conjunction with a membranetranslocating polypeptide. In some embodiments, the membranetranslocating polypeptide is a targeting moiety. In some embodiments,the membrane translocating polypeptide is a delivery agent that aidsdelivery of the targeting moiety described herein. The target locationmay be intracellular, e.g., cytosolic or intra-organellar (e.g.,intranuclear, such as a target DNA sequence or chromatin structure). Thetherapeutic compositions described herein may have further advantageousproperties, such as improved targeting, absorption, or transport, orreduced off-target activity, toxic metabolism, or toxic excretion.

In one embodiment, the composition includes at least one membranetranslocating polypeptide with each comprising at least one sequence ofABX^(n)C, where A is selected from a hydrophobic amino acid or an amidecontaining backbone, e.g., aminoethyl-glycine, with a nucleic acid sidechain; B and C may be the same or different, and are independentlyselected from arginine, asparagine, glutamine, lysine, and analogsthereof; X is each independently a hydrophobic amino acid or X is eachindependently an amide containing backbone, e.g., aminoethyl-glycine,with a nucleic acid side chain; and n is an integer from 1 to 4.

Hydrophobic amino acids include amino acids having hydrophobic sidechains and include, but are not limited to, alanine (ala, A), valine(val, V), isoleucine (iso, I), leucine (leu, L), methionine (met, M),phenylalanine (phe, F), tyrosine (tyr, Y), tryptophan (trp, W), andanalogs thereof.

Amino acid analogs include, but are not limited to, D-amino acids, aminoacids lacking a hydrogen on the α-carbon such as dehydroalanine,metabolic intermediates such as ornithine and citrulline, non-alphaamino acids such as β-alanine, γ-aminobutyric acid, and 4-aminobenzoicacid, twin α-carbon amino acids such as cystathionine, lanthionine,djenkolic acid and diaminopimelic acid, and any others known in the art.

Nucleic Acid Side Chains

In one embodiment, the membrane translocating polypeptide includes oneor more nucleic acid side chains linked to the amide backbone. Anindividual amino acid unit in a polypeptide includes the amide bond andits corresponding side chain. One or more amino acid units in themembrane translocating polypeptide have an amide containing backbone,e.g., aminoethyl-glycine, similar to a peptide backbone, with a nucleicacid side chain in place of the amino acid side chain. Peptide nucleicacids (PNA) are known to hybridize complementary DNA and RNA with higheraffinity than their oligonucleotide counterparts. This character of PNAnot only makes the polypeptide of the disclosure a stable hybrid withthe nucleic acid side chains, but at the same time, the neutral backboneand hydrophobic side chains result in a hydrophobic unit within thepolypeptide.

The nucleic acid side chain includes, but is not limited to, a purine ora pyrimidine side chain such as adenine, cytosine, guanine, thymine anduracil. In one embodiment, the nucleic acid side chain includes anucleoside analog as described herein.

Size

In some embodiments, the membrane translocating polypeptide has a sizein the range of about 5 to about 500, e.g., 5-400, 5-300, 5-250, 5-200,5-150, 5-100 amino acid units in length. The polypeptide may have alength in the range of about 5 to about 50 amino acids, about 5 to about40 amino acids, about 5 to about 30 amino acids, about 5 to about 25amino acids, or any other range. In one embodiment, the polypeptide hasa length of about 10 amino acids. In another embodiment, the polypeptidehas a length of about 15 amino acids. In another embodiment, thepolypeptide has a length of about 20 amino acids. In another embodiment,the polypeptide has a length of about 25 amino acids. In anotherembodiment, the polypeptide has a length of about 30 amino acids.

The membrane translocating polypeptide may have more than one sequenceof ABX^(n)C within its length. Each ABX^(n)C sequence may be separatedfrom another ABX^(n)C sequence by one or more amino acids. In oneembodiment, the polypeptide repeats the ABX^(n)C sequence and separatesthe sequences by one or more amino acid units. In another embodiment,the polypeptide includes at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15 or more, e.g., between 2-20, between 2-10, between2-5) ABX^(n)C sequences and separates the sequences by one or more aminoacid units. In another embodiment, the ABX^(n)C sequences are separatedby one (or more) hydrophobic amino acid, such as isoleucine or leucine.

The composition may include a plurality of ABX^(n)C sequences that arethe same or different. In one embodiment, at least two of the pluralityare identical in sequence and/or length. In one embodiment, at least twoof the plurality are different in sequence and/or length. In oneembodiment, the composition includes a plurality of ABX^(n)C sequenceswherein at least two of the plurality are the same and at least 2 of theplurality are different. In one embodiment, the ABX^(n)C sequences inthe membrane translocating polypeptide are not identical in sequence orlength or a combination thereof.

Production of Proteins or Polypeptides

Methods of making the therapeutic protein or polypeptide describedherein are routine in the art. See, in general, Smales & James (Eds.),Therapeutic Proteins: Methods and Protocols (Methods in MolecularBiology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (Eds.),Pharmaceutical Biotechnology: Fundamentals and Applications, Springer(2013).

The protein or polypeptide of the composition can be biochemicallysynthesized by employing standard solid phase techniques. Such methodsinclude exclusive solid phase synthesis, partial solid phase synthesismethods, fragment condensation, classical solution synthesis. Thesemethods can be used when the peptide is relatively short (i.e., 10 kDa)and/or when it cannot be produced by recombinant techniques (i.e., notencoded by a nucleic acid sequence) and therefore involves differentchemistry.

Solid phase synthesis procedures are well known in the art and furtherdescribed by John Morrow Stewart and Janis Dillaha Young, Solid PhasePeptide Syntheses, 2nd Ed., Pierce Chemical Company, 1984; and Coin, I.,et al., Nature Protocols, 2:3247-3256, 2007.

For longer peptides, recombinant methods may be used. Methods of makinga recombinant therapeutic polypeptide are routine in the art. See, ingeneral, Smales & James (Eds.), Therapeutic Proteins: Methods andProtocols (Methods in Molecular Biology), Humana Press (2005); andCrommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology:Fundamentals and Applications, Springer (2013).

Exemplary methods for producing a therapeutic pharmaceutical protein orpolypeptide involve expression in mammalian cells, although recombinantproteins can also be produced using insect cells, yeast, bacteria, orother cells under the control of appropriate promoters. Mammalianexpression vectors may comprise nontranscribed elements such as anorigin of replication, a suitable promoter, and other 5′ or 3′ flankingnontranscribed sequences, and 5′ or 3′ nontranslated sequences such asnecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, and termination sequences. DNA sequences derivedfrom the SV40 viral genome, for example, SV40 origin, early promoter,splice, and polyadenylation sites may be used to provide the othergenetic elements required for expression of a heterologous DNA sequence.Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts are described in Green &Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), ColdSpring Harbor Laboratory Press (2012).

In cases where large amounts of the protein or polypeptide are desired,it can be generated using techniques such as described by Brian Bray,Nature Reviews Drug Discovery, 2:587-593, 2003; and Weissbach &Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press,NY, Section VIII, pp 421-463.

Various mammalian cell culture systems can be employed to express andmanufacture recombinant protein. Examples of mammalian expressionsystems include CHO cells, COS cells, HeLA and BHK cell lines. Processesof host cell culture for production of protein therapeutics aredescribed in Zhou and Kantardjieff (Eds.), Mammalian Cell Cultures forBiologics Manufacturing (Advances in BiochemicalEngineering/Biotechnology), Springer (2014). The compositions describedherein may include a vector, such as a viral vector, e.g., a lentiviralvector, encoding the recombinant protein. The vector, e.g., a viralvector, that comprises the nucleic acid encoding the recombinantprotein.

Purification of protein therapeutics is described in Franks, ProteinBiotechnology: Isolation, Characterization, and Stabilization, HumanaPress (2013); and in Cutler, Protein Purification Protocols (Methods inMolecular Biology), Humana Press (2010).

Formulation of protein therapeutics is described in Meyer (Ed.),Therapeutic Protein Drug Products: Practical Approaches to formulationin the Laboratory, Manufacturing, and the Clinic, Woodhead PublishingSeries (2012).

Linkers

The proteins or polypeptides describe herein may also include a linker.In some embodiments, the protein described herein, e.g., comprising afirst polypeptide domain that comprises a Cas or modified Cas proteinand a second polypeptide domain that comprises a polypeptide having DNAmethyltransferase activity [or associated with demethylation ordeaminase activity], has a linker between the first and secondpolypeptide. In one embodiment, one or more polypeptides describedherein are linked with a linker. A linker may be a chemical bond, e.g.,one or more covalent bonds or non-covalent bonds. In some embodiments,the linker is a peptide linker (e.g., a non ABX^(n)C peptide). Such alinker may be between 2-30 amino acids, or longer. The linker includesflexible, rigid or cleavable linkers described herein.

The most commonly used flexible linkers have sequences consistingprimarily of stretches of Gly and Ser residues (“GS” linker). Flexiblelinkers may be useful for joining domains that require a certain degreeof movement or interaction and may include small, non-polar (e.g. Gly)or polar (e.g. Ser or Thr) amino acids. Incorporation of Ser or Thr canalso maintain the stability of the linker in aqueous solutions byforming hydrogen bonds with the water molecules, and therefore reduceunfavorable interactions between the linker and the protein moieties.

Rigid linkers are useful to keep a fixed distance between domains and tomaintain their independent functions. Rigid linkers may also be usefulwhen a spatial separation of the domains is critical to preserve thestability or bioactivity of one or more components in the fusion. Rigidlinkers may have an alpha helix-structure or Pro-rich sequence, (XP)₁₁,with X designating any amino acid, preferably Ala, Lys, or Glu.

Cleavable linkers may release free functional domains in vivo. In someembodiments, linkers may be cleaved under specific conditions, such asthe presence of reducing reagents or proteases. In vivo cleavablelinkers may utilize the reversible nature of a disulfide bond. Oneexample includes a thrombin-sensitive sequence (e.g., PRS) between thetwo Cys residues. In vitro thrombin treatment of CPRSC results in thecleavage of the thrombin-sensitive sequence, while the reversibledisulfide linkage remains intact. Such linkers are known and described,e.g., in Chen et al. 2013. Fusion Protein Linkers: Property, Design andFunctionality. Adv Drug Deliv Rev. 65(10): 1357-1369. In vivo cleavageof linkers in fusions may also be carried out by proteases that areexpressed in vivo under pathological conditions (e.g. cancer orinflammation), in specific cells or tissues, or constrained withincertain cellular compartments. The specificity of many proteases offersslower cleavage of the linker in constrained compartments.

Examples of linking molecules include a hydrophobic linker, such as anegatively charged sulfonate group; lipids, such as a poly (—CH₂—)hydrocarbon chains, such as polyethylene glycol (PEG) group, unsaturatedvariants thereof, hydroxylated variants thereof, amidated or otherwiseN-containing variants thereof, noncarbon linkers; carbohydrate linkers;phosphodiester linkers, or other molecule capable of covalently linkingtwo or more polypeptides. Non-covalent linkers are also included, suchas hydrophobic lipid globules to which the polypeptide is linked, forexample through a hydrophobic region of the polypeptide or a hydrophobicextension of the polypeptide, such as a series of residues rich inleucine, isoleucine, valine, or perhaps also alanine, phenylalanine, oreven tyrosine, methionine, glycine or other hydrophobic residue. Thepolypeptide may be linked using charge-based chemistry, such that apositively charged moiety of the polypeptide is linked to a negativecharge of another polypeptide or nucleic acid.

Multimerization of Polypeptides

The composition may include a plurality (two or more) of membranetranslocating polypeptides linked together, e.g., through a linkerdescribed herein.

The composition may include a plurality of membrane translocatingpolypeptides that are the same or different. In one embodiment, at leasttwo of the plurality are identical in sequence and/or length. In oneembodiment, at least two of the plurality are different in sequenceand/or length. In one embodiment, the composition includes a pluralityof polypeptides wherein at least two of the plurality are the same andat least 2 of the plurality are different. In one embodiment, thepolypeptides in the composition are not identical in sequence or lengthor a combination thereof.

The composition includes a membrane translocating polypeptide that islinked to another membrane translocating polypeptide, e.g., by a linker.In some embodiments, the composition includes two or more polypeptideslinked by a linker. In some embodiments, the composition includes threeor more polypeptides linked by linkers. In some embodiments, thecomposition includes four or more polypeptides linked by linkers. Insome embodiments, the composition includes five or more polypeptideslinked by linkers. The linker may be a chemical bond, e.g., one or morecovalent bonds or non-covalent bonds, e.g., a flexible, rigid orcleavable peptide linker. Such a linker may be between 2-30 amino acids,or longer. Additional linkers are described in more detail elsewhereherein and are also applicable.

In one embodiment, two or more membrane translocating polypeptides arelinked through a peptide bond, for example the carboxyl terminal of onepolypeptide is bonded to the amino terminal of another polypeptide. Inanother embodiment, one or more amino acids on one polypeptide arelinked with one or more amino acids on another polypeptide, such asthrough disulfide bonds between cysteine side chains. In anotherembodiment, one or more amino acids on one polypeptide are linked with acarboxyl or amino terminal on another polypeptide, such as to create abranched polypeptide.

In another embodiment, one or more nucleic acid side chains on onemembrane translocating polypeptide interact with one or more amino acidside chains on another membrane translocating polypeptide, such asthrough arginine forming a pseudo-pairing with guanosine. In anotherembodiment, one or more nucleic acid side chains on one membranetranslocating polypeptide interact with one or more nucleic acid sidechains on another membrane translocating polypeptide, such as throughhydrogen bonding. In another embodiment, multiple membrane translocatingpolypeptides interact to create a specific sequence in the arrangementof the nucleic acid side chains. For example, the carboxy terminalnucleic acid side chain from one polypeptide interacts with the aminoterminal nucleic acid side chain from another polypeptide to create apseudo-5′ to pseudo-3′ nucleotide sequence. In another example, apolypeptide is linked with one or more polypeptides, such as throughamino acids and/or terminus on each polypeptide, and their respectivenucleic acid side chains align to create a pseudo-5′ to pseudo-3′nucleotide sequence. The pseudo-sequence may bind a selected targetsequence, such as an anchor sequence of an anchor sequence-mediatedconjunction, e.g., a CTCF binding motif, cohesin binding mofitf, USF1binding motif, YY1 binding motif, TATA-box, ZNF143 binding motif, etc.,or a transcriptional control sequence, e.g., an enhancing or silencingsequence. The pseudo-sequence may bind a selected target sequence, suchas a transcriptional control sequence, e.g., an enhancing or silencingsequence. The pseudo-sequence may interfere with factor binding andtranscription by binding to a target sequence. The pseudo-sequence mayhybridize with a nucleic acid sequence, such as an mRNA to interferewith gene expression.

In one embodiment, the membrane translocating polypeptides are linked toone another and the linked polypeptides create a pseudo-5′ to pseudo-3′nucleotide sequence that binds to an anchor sequence that is recognizedby a nucleating protein that binds with sufficient avidity to form ananchor sequence-mediated conjunction, e.g., a loop, or a two-dimensionalDNA structure generated by the physical interaction or binding of oneconjunction nucleating molecule-anchor sequence with another conjunctionnucleating molecule-anchor sequence. An example of an anchor sequenceincludes, but is not limited to, a CTCF binding motif, e.g.,CTCF-binding motif or consensus sequence:N(T/C/G)N(G/A/T)CC(A/T/G)(C/G)(C/T/A)AG(G/A)(G/T)GG(C/A/T)(G/A)(C/G)(C/T/A)(G/A/C)(SEQ ID NO:1), where N is any nucleotide. The linked polypeptides maycreate a pseudo-5′ to pseudo-3′ nucleotide sequence that binds to aCTCF-binding motif or consensus sequence in the opposite orientation,e.g.,(G/A/C)(C/T/A)(C/G)(G/A)(C/A/T)GG(G/T)(G/A)GA(C/T/A)(C/G)(A/T/G)CC(G/A/T)N(T/C/G)N(SEQ ID NO:2).

The membrane translocating polypeptides described herein can bemultimerized, e.g., linking two or more polypeptides, by employingstandard ligation techniques. Such methods include, general nativechemical ligation strategies (Siman, P. and Brik, A. Org. Biomol. Chem.2012, 10:5684-5697; Kent, S. B. H. Chem. Soc. Rev. 2009, 38:338-351; andHackenberger, C. P. R. and Schwarzer, D. Angew. Chem., Int. Ed. 2008,47:10030-10074), click modification protocols (Tasdelen, M. A.; Yagci,Y. Angew. Chem., Int. Ed. 2013, 52:5930-5938; Palomo, J. M. Org. Biomol.Chem. 2012, 10:9309-9318; Eldijk, M. B.; van Hest, J. C. M. Angew.Chem., Int. Ed. 2011, 50:8806-8827; and Lallana, E.; Riguera, R.;Fernandez-Megia, E. Angew. Chem., Int. Ed. 2011, 50:8794-8804), andbioorthogonal reactions (King, M.; Wagner, A. Bioconjugate Chem. 2014,25:825-839; Lang, K.; Chin, J. W. Chem. Rev. 2014, 114:4764-4806;Patterson, D. M.; Nazarova, L. A.; Prescher, J. A. ACS Chem. Biol. 2014,9:592-605; Lang, K.; Chin, J. W. ACS Chem. Biol. 2014, 9:16-20; akaoka,Y.; Ojida, A.; Hamachi, I. Angew. Chem., Int. Ed. 2013, 52:4088-4106;Debets, M. F.; van Hest, J. C. M.; Rutjes, F. P. J. T. Org. Biomol.Chem. 2013, 11:6439-6455; and Ramil, C. P.; Lin, Q. Chem. Commun. 2013,49:11007-11022).

In some embodiments, the ordering of the membrane translocatingpolypeptides in the multimer is specific or it may be random, e.g., whenthe polypeptides are not identical. For example, the polypeptidesdescribed herein are multimerized by template driven synthesis ormultimerization is ordered by physical constraints or hybridization to atemplate, e.g., DNA, protein, hybrid DNA-protein. In one embodiment, atemplate, e.g., a DNA sequence, specifically hybridizes to a polypeptidedescribed herein. The polypeptide is linked to another polypeptide viaone of the methods described herein, e.g., general chemical ligation,and the choice of which polypeptide is linked may be constrained by theability to hybridize to the template. Thus, a specific polypeptidemultimer may be generated by its ability to specifically hybridize tothe template.

In some embodiments, the order of the membrane translocatingpolypeptides in the multimer is determined by the chemical ligationstrategy used. In one embodiment, chemical ligation techniques, such asclick chemistry and bioorthogonal reactions, direct which polypeptidesare linked because the chemical ligation strategy requires specificentities to react for the ligation technique to proceed. For example,one polypeptide may be labeled with a phenyl azide and anotherpolypeptide is labeled with cyclooctyne. The cyclooctyne and phenylazide react to link the two polypeptides.

Hybridization

In embodiments where the membrane translocating polypeptide includesnucleic acid side chains, it is capable of interacting with nucleicacids. In one embodiment, one or more nucleic acid side chains on thepolypeptide hybridize with a nucleic acid sequence, e.g., a DNA such asgenomic DNA, RNA such as siRNA or mRNA molecule. One or more of thenucleic acid side chains on the polypeptide specifically hybridizes withone or more nucleic acid residues in a target nucleic acid sequence. Inone embodiment, the polypeptides are linked to one another and thenucleic acid side chains hybridize a nucleic acid sequence (e.g., genelocus, mRNA, anchor sequence of an anchor sequence-mediated conjunction,e.g., CTCF binding motif, cohesin binding motif, USF1 binding motif, YY1binding motif, TATA-box, ZNF143 binding motif, etc.).

The nucleic acid side chains or pseudo-sequence of nucleic acid sidechains may hybridize a target nucleic acid sequence that issubstantially matched to hybridize or 100%, 95%, 90%, 85%, 80%, 75%, or70% complementary to the nucleic acid side chains or pseudo-sequence ofnucleic acid side chains. Hybridization of the nucleic acid side chainsor pseudo-sequence of nucleic acid side chains with a target nucleicacid sequence may be carried out under suitable hybridization conditionsroutinely determined by optimization procedures. Conditions such astemperature, concentration of components, hybridization and washingtimes, buffer components, and their pH and ionic strength may be varieddepending on various factors, including the length and GC content ofnucleic acid side chains or pseudo-sequence of nucleic acid side chainsand the complementary target nucleic acid sequence. For example, when arelatively short length of nucleic acid side chains or pseudo-sequenceof nucleic acid side chains is used, lower stringent conditions may beadopted. The detailed conditions for hybridization can be found inMolecular Cloning, A laboratory manual, fourth edition (Cold SpringHarbor Laboratory Press, 2012) or the like.

Polypeptide Linked Heterologous Moiety

The composition may include a heterologous moiety described hereinlinked to the membrane translocating polypeptide of the targetingmoiety, such as through covalent bonds or non-covalent bonds or a linkeras described herein. In one embodiment, the composition comprises aheterologous moiety linked to the polypeptide through a peptide bond.For example, the amino terminal of the polypeptide is linked to theheterologous moiety, such as through a peptide bond with an optionallinker. In another embodiment, the carboxyl terminal of the polypeptideis linked to the heterologous moiety.

In one embodiment, the composition comprises a membrane translocatingpolypeptide linked to two heterologous moieties. For example, the aminoterminal and carboxyl terminal of the polypeptide are linked toheterologous moieties, which may be the same or different heterologousmoieties.

In another embodiment, one or more amino acids of the membranetranslocating polypeptide are linked with the heterologous moiety, suchas through disulfide bonds between cysteine side chains, hydrogenbonding, or any other known chemistry. One heterologous moiety may be aneffector with biological activity and the other heterologous moiety maybe a ligand or antibody to target the composition to a specific cellexpressing the receptor. For example, a chemotherapeutic agent, such astopotecan a topoisomerase inhibitor, is linked to one end of thepolypeptide and a ligand or antibody is linked to the other end of thepolypeptide to target the composition to a specific cell or tissue. Inanother example, the heterologous moieties are both effectors withbiological activity.

In another embodiment, a plurality of membrane translocatingpolypeptides, either the same or different membrane translocatingpolypeptides, are linked to a single heterologous moiety. Thepolypeptides may act as a coating that surrounds a large heterologousmoiety and aids in its membrane penetration. The heterologous moiety mayhave a molecular weight greater than about 500 grams per mole ordaltons, e.g., organic or inorganic compound has a molecular weightgreater than about 1,000 grams per mole, e.g., organic or inorganiccompound has a molecular weight greater than about 2,000 grams per mole,e.g., organic or inorganic compound has a molecular weight greater thanabout 3,000 grams per mole, e.g., organic or inorganic compound has amolecular weight greater than about 4,000 grams per mole, e.g., organicor inorganic compound has a molecular weight greater than about 5,000grams per mole, and salts, esters, and other pharmaceutically acceptableforms of such compounds are included.

In one embodiment, the composition comprises a membrane translocatingpolypeptide linked to a heterologous moiety on one or both ends andanother heterologous moiety linked to another site on the polypeptide.One or both the amino terminal and the carboxyl terminal of thepolypeptide is linked to the heterologous moiety and one or more aminoacid units in the polypeptide, either amino acids or nucleic acids, islinked to one or more heterologous moieties, such as through disulfidebonds or hydrogen bonding. For example, a DNA modification enzyme islinked to the polypeptide, and a nucleic acid having an unmethylatedCTCF binding motif that is complementary to a target methylated gene ishybridized to the nucleic acid side chains of the polypeptide. Uponadministration, the composition targets the CTCF genomic binding motifto modulate transcription of the gene. In another example, a doublestranded nucleic acid having an unmethylated CTCF binding motif withgene specific flanking sequences is linked to the polypeptide. Uponadministration, the unmethylated CTCF binding motif serves as analternate anchor sequence for CTCF protein to bind. In another example,ubiquitin and another heterologous moiety, such as an effector, arelinked to the polypeptide. Upon administration, the compositionpenetrates the cell membrane and the effector performs a function. Then,ubiquitin targets the composition for degradation.

In one embodiment, the composition comprises a membrane translocatingpolypeptide linked to one or more heterologous moieties through covalentbonds and another heterologous moiety linked to the nucleic acids in thepolypeptide. For example, a protein synthesis inhibitor is covalentlylinked to the polypeptide, and an siRNA or other target specific nucleicacid is hybridized to the nucleic acids in the polypeptide. Uponadministration, the siRNA targets the composition to an mRNA transcriptand the protein synthesis inhibitor and siRNA act to inhibit expressionof the mRNA.

In some embodiments, the pharmaceutical composition comprises a membranetranslocating polypeptide linked to a gRNA that comprises a sequence ofstructure I:

X—Y—Z,  (II)

-   -   where X and Z are 5′ and 3′ site specific targeting sequences        for a target CTCF binding motif, respectively, and Y is selected        from:    -   (a) an RNA sequence complementary to the sequence of SEQ ID        NO:1;    -   (b) an RNA sequence at least 75%, 80%, 85%, 90%, 95% identical        to an RNA sequence complementary to the sequence of SEQ ID NO:1;    -   (c) an RNA sequence complementary to the sequence of SEQ ID NO:1        having at least 1, 2, 3, 4, 5, but less than 15, 12 or 10        nucleotide additions, substitutions or deletions.    -   (d) an RNA sequence complementary to the sequence of SEQ ID        NO:2;    -   (e) an RNA sequence at least 75%, 80%, 85%, 90%, 95% identical        to an RNA sequence complementary to the sequence of SEQ ID NO:2;    -   (f) an RNA sequence complementary to the sequence of SEQ ID NO:2        having at least 1, 2, 3, 4, 5, but less than 15, 12 or 10        nucleotide additions, substitutions or deletions.

In some embodiments, X and Z are each between 2-50 nucleotides inlength, e.g., between 2-20, between 2-10, between 2-5 nucleotides inlength.

In some embodiments, a gRNA comprises a specific targeting sequence fora CTCF binding motif associated with an oncogene, a tumor suppressor, ora disease associated with a nucleotide repeat.

The membrane translocating polypeptides described herein can be linkedto a heterologous moiety by employing standard ligation techniques, suchas those described herein to link polypeptides.

For introducing small mutations or a single-point mutation, a homologousrecombination (HR) template can be linked to the membrane translocatingpolypeptide. In one embodiment, the HR template is a single stranded DNA(ssDNA) oligo or a plasmid. For ssDNA oligo design, one may use around100-150 bp total homology with the mutation introduced roughly in themiddle, giving 50-75 bp homology arms.

In some embodiments, a gRNA or antisense DNA oligonucleotide fortargeting a target anchor sequence, e.g., a CTCF binding motif, islinked to the membrane translocating polypeptide in combination with anHR template selected from:

-   -   (a) a nucleotide sequence comprising SEQ ID NO:1;    -   (b) a nucleotide sequence at least 75%, 80%, 85%, 90%, 95%        identical to SEQ ID NO:1;    -   (c) a nucleotide sequence comprising SEQ ID NO:1 having at least        1, 2, 3, 4, 5, but less than 15, 12 or 10 nucleotide additions,        substitutions or deletions.    -   (d) a nucleotide sequence comprising SEQ ID NO:2;    -   (e) a nucleotide sequence at least 75%, 80%, 85%, 90%, 95%        identical to SEQ ID NO:2;    -   a nucleotide sequence comprising SEQ ID NO:2 having at least 1,        2, 3, 4, 5, but less than 15, 12 or 10 nucleotide additions,        substitutions or deletions.

Any of the linkers described herein may be included to covalently ornoncovalently link the membrane translocating polypeptide and aheterologous moiety. The linker can be used, e.g., to space thepolypeptide from the heterologous moiety. For example, the linker can bepositioned between the polypeptide and the heterologous moiety, e.g., toprovide molecular flexibility of secondary and tertiary structures. Inone embodiment, the linker includes at least one glycine, alanine, andserine amino acids to provide for flexibility. In another embodiment,the linker is a hydrophobic linker, such as including a negativelycharged sulfonate group, polyethylene glycol (PEG) group, orpyrophosphate diester group. In another embodiment, the linker iscleavable to selectively release the heterologous moiety from thepolypeptide, but sufficiently stable to prevent premature cleavage.

Linkage after Administration

In some embodiments, the membrane translocating polypeptide describedherein has the capacity to form linkages, e.g., after administration, toother polypeptides, to a heterologous moiety as described herein, e.g.,an effector molecule, e.g., a nucleic acid, protein, peptide or othermolecule, or other agents, e.g., intracellular molecules, such asthrough covalent bonds or non-covalent bonds. In one embodiment, one ormore amino acids on the polypeptide are capable of linking with anucleic acid, such as through arginine forming a pseudo-pairing withguanosine or an internucleotide phosphate linkage or an interpolymericlinkage. In some embodiments, the nucleic acid is a DNA such as genomicDNA, RNA such as tRNA or mRNA molecule. In another embodiment, one ormore amino acids on the polypeptide are capable of linking with aprotein or peptide.

Fusion Molecules

In some embodiments, the composition comprises a fusion molecule, suchas a fusion molecule that comprises a peptide or polypeptide. Thoseskilled in the art reading the specification would appreciate that theterm “protein fusion” may refer to a fusion molecule that comprises a“protein” (or peptide or polypeptide) component. In some embodiments,the protein fusion comprises one or more of the moieties describedherein, e.g., a nucleic acid sequence, a peptide or protein moiety, amembrane translocating polypeptide, a targeting peptide/aptamer, orother heterologous moiety described herein.

In one aspect, the disclosure includes a cell or tissue comprising anyone of the protein fusions described herein.

In another aspect, the disclosure includes a pharmaceutical compositioncomprising the protein fusion described herein.

In another aspect, the disclosure includes a method of modulatingexpression of a gene by administering the composition comprising theprotein fusion described herein. For example, the protein fusion may bedCas9-DNMT, dCas9-DNMT-3a-3L, dCas9-DNMT-3a-3a, dCas9-DNMT-3a-3L-3a,dCas9-DNMT-3a-3L-KRAB, dCas9-KRAB, dCas9-APOBEC, APOBEC-dCas9,dCas9-APOBEC-UGI, dCas9-UGI, UGI-dCas9-APOBEC, UGI-APOBEC-dCas9, anyvariation of the protein fusions described herein, or other fusions ofproteins or protein domains described herein.

Exemplary dCas9 fusion methods and compositions that are adaptable tothe methods and compositions described herein are known and aredescribed, e.g., in Kearns et al., Functional annotation of nativeenhancers with a Cas9-histone demethylase fusion. Nature Methods 12,401-403 (2015); and McDonald et al., Reprogrammable CRISPR/Cas9-basedsystem for inducing site-specific DNA methylation. Biology Open 2016:doi: 10.1242/bio.019067. Using methods known in the art, dCas9 can befused to any of a variety of agents and/or molecules as describedherein; such resulting fusion molecules can be useful in variousdisclosed methods.

In one aspect, the disclosure includes a composition comprising aprotein comprising a domain, e.g., an enzyme domain, that acts on DNA(e.g., a nuclease domain, e.g., a Cas9 domain, e.g., a dCas9 domain; aDNA methyltransferase, a demethylase, a deaminase), in combination withat least one guide RNA (gRNA) or antisense DNA oligonucleotide thattargets the protein to an anchor sequence of a target anchorsequence-mediated conjunction,

wherein the composition is effective to alter, in a human cell, thetarget anchor sequence-mediated conjunction. In some embodiments, theenzyme domain is a Cas9 or a dCas9. In some embodiments, the proteincomprises two enzyme domains, e.g., a dCas9 and a methylase ordemethylase domain.

In some embodiments, the targeting moiety includes a fusion of asequence targeting polypeptide and a conjunction nucleating molecule,e.g. a fusion of dCas9 and a conjunction nucleating molecule, e.g., onegRNA or antisense DNA oligonucleotides fused with a nuclease, or anucleic acid encoding the fusion. Fusions of a catalytically inactiveendonuclease e.g., a dead Cas9 (dCas9, e.g., D10A; H840A) tethered withall or a portion of (e.g., biologically active portion of) an (one ormore) effector domain and/or other agent create chimeric proteins orfusion molecules that can be guided to specific DNA sites by one or moreRNA sequences (sgRNA) or antisense DNA oligonucleotides to modulateactivity and/or expression of one or more target nucleic acids sequences(e.g., to methylate or demethylate a DNA sequence).

As used herein, a “biologically active portion of an effector domain” isa portion that maintains the function (e.g. completely, partially,minimally) of an effector domain (e.g., a “minimal” or “core” domain).In some embodiments, fusion of a dCas9 with all or a portion of one ormore effector domains of an epigenetic modifying agent (such as a DNAmethylase or enzyme with a role in DNA demethylation, e.g., DNMT3a,DNMT3b, DNMT3L, a DNMT inhibitor, TET family enzymes, and combinationsthereof, or protein acetyl transferase or deacetylase) creates achimeric protein that is useful in the methods described herein.Accordingly, in some embodiments, the targeting moiety includes adCas9-methylase fusion in combination with a site-specific gRNA orantisense DNA oligonucleotide that targets the fusion to a conjunctionanchor sequence (such as a CTCF binding motif), thereby decreasing theaffinity or ability of the anchor sequence to bind a conjunctionnucleating protein. In other some embodiments, the targeting moietyincludes a dCas9-enzyme fusion in combination with a site-specific gRNAor antisense DNA oligonucleotide that targets the fusion to aconjunction anchor sequence (such as a CTCF binding motif), therebyincreasing the affinity or ability of the anchor sequence to bind aconjunction nucleating molecule. In some embodiments, all or a portionof one or more epigenetic modifying agent effector domains (e.g., DNAmethylase or enzyme with a role in DNA demethylation, or protein acetyltransferase or deacetylase, or deaminase) are fused with the inactivenuclease, e.g., dCas9. In other aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more effector domains (all ora biologically active portion) are fused with dCas9.

The chimeric proteins described herein may also comprise a linker, e.g.,an amino acid linker. In some aspects, a linker comprises 2 or moreamino acids, e.g., one or more GS sequences. In some aspects, fusion ofCas9 (e.g., dCas9) with two or more effector domains (e.g., of a DNAmethylase or enzyme with a role in DNA demethylation or protein acetyltransferase or deacetylase) comprises one or more interspersed linkers(e.g., GS linkers) between the domains. In some aspects, dCas9 is fusedwith 2-5 effector domains with interspersed linkers.

Modifying Chromatin Structure

The methods described herein modulate chromatin structure (e.g., anchorsequence-mediated conjunctions) in order to modulate gene expression ina subject, e.g., by modifying anchor sequence-mediated conjunctions inDNA. Those skilled in the art reading the present specification willappreciate that modulations described herein may modulate chromatinstructure in a way that would alter its two-dimensional representation(e.g., would add, alter, or delete a loop or other anchorsequence-mediated conjunction); such modulations are referred to herein,in accordance with common parlance, as modulations or modification of atwo-dimensional structure.

In one aspect, the methods described herein may comprise modifying atwo-dimensional structure by altering a topology of an anchorsequence-mediated conjunction, e.g., a loop, to modulate transcriptionof a nucleic acid sequence, wherein the altered topology of the anchorsequence-mediated conjunction modulates transcription of the nucleicacid sequence.

In another aspect, the methods described herein may comprise modifying atwo-dimensional structure chromatin structure by altering a topology ofa plurality of anchor sequence-mediated conjunctions, e.g., multipleloops, to modulate transcription of a nucleic acid sequence, wherein thealtered topology modulates transcription of the nucleic acid sequence.

In another aspect, the methods described herein may comprise modulatingtranscription of a nucleic acid sequence by altering an anchorsequence-mediated conjunction, e.g., a loop, that influencestranscription of a nucleic acid sequence, wherein altering the anchorsequence-mediated conjunction modulates transcription of the nucleicacid sequence.

In some embodiments, altering the anchor sequence-mediated conjunctioncomprises modifying a chromatin structure, e.g., disrupting [reversibleor irreversible] a topology of the anchor sequence-mediated conjunction,altering one or more nucleotides in the anchor sequence-mediatedconjunction [genetically modifying the sequence], epigeneticallymodifying [modulating DNA methylation at one or more sites] the anchorsequence-mediated conjunction, or forming a non-naturally occurringanchor sequence-mediated conjunction. In some embodiments, altering theanchor sequence-mediated conjunction comprises modifying a chromatinstructure.

As appreciated by those of skill in the art, a given pair of anchorsequences may “breathe” in and out of an anchor sequence-mediatedconjunction, though a given pair of anchor sequences may tend to be moreor less often in a particular state (either in or out of a conjunction)depending on factors, such as, for example, cell type.

By “disruption” it is meant that formation and/or stability of an anchorsequence-mediated conjunction is negatively affected.

Reversible Disruption

In some embodiments, compositions and methods are described herein forreversibly disrupting an anchor sequence-mediated conjunction. Forexample, the disruption may transiently modulate transcription, e.g., amodulation that persists for no more than about 30 mins to about 7 days,or no more than about 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17hrs, 18 hrs, 19 hrs, 20 hrs, 21 hrs, 22 hrs, 24 hrs, 36 hrs, 48 hrs, 60hrs, 72 hrs, 4 days, 5 days, 6 days, 7 days, or any time therebetween.

In some embodiments, a targeting moiety described herein interferes withloop formation by, e.g., CTCF and CTCF-binding motif by blocking theinteraction between CTCF and the CTCF-binding motif. In one embodiment,a composition or method is described for disrupting an anchorsequence-mediated conjunction with an epigenetic modifying agent, suchas a gRNA, that targets DNA and acts as a steric presence in thevicinity of the anchoring sequence. The gRNA recognizes specific DNAsequences (e.g., an anchor sequence, a CTCF anchor sequence, flanked bysequences that confer sequence specificity). The gRNA may includeadditional sequences that interfere with a conjunction nucleatingmolecule sequence to act as a steric blocker. In some embodiments, thegRNA is combined with one or more peptides, e.g., S-adenosyl methionine(SAM), to act as a steric presence to interfere with a conjunctionnucleating molecule. Degradation of the gRNA removes the stericpresence, thereby allowing the conjunction nucleating molecule to gainaccess to the conjunction nucleating molecule sequence.

In some embodiments, a targeted alteration described herein reversiblydisrupts the anchor sequence-mediated conjunction. In one embodiment, acomposition or method is described for modifying an anchorsequence-mediated conjunction with an epigenetic modifying agent, suchas a gene editing system, to target DNA in the vicinity of the anchoringsequence. gRNA recognizes specific DNA sequences (e.g., an anchorsequence, a CTCF anchor sequence, flanked by sequences that confersequence specificity) and nuclease-deficient Cas9 recruits transcriptionrepressors, e.g., to induce epigenetic modifications in the vicinity ofthe anchoring sequence. Transcription activators, e.g., may beselectively recruited to reverse the epigenetic modification made by thetranscription repressors.

In another embodiment, a composition or method is described forintroducing an exogenous anchor sequence to alter an anchorsequence-mediated conjunction. A non-naturally occurring or exogenousanchor sequence is introduced that forms a non-naturally occurring loopor disrupts a naturally occurring anchor sequence-mediated conjunctionto form that alters transcription of the nucleic acid sequence. Removalof the exogenous anchor sequence prevents formation of the non-naturallyoccurring loop or the reformation of the naturally occurring anchorsequence-mediated conjunction.

In some embodiments, the binding affinity of a conjunction nucleatingmolecule is altered, e.g., for an anchor sequence within the anchorsequence-mediated conjunction, an alternative splicing site, or abinding site for a non-translated RNA. In one embodiment, a compositionor method is described for disrupting an anchor sequence-mediatedconjunction with an engineered conjunction nucleating molecule withaltered binding affinity, e.g. conjunction nucleating molecule disrupts,e.g., by competitive binding, the binding of an endogenous conjunctionnucleating molecule to its binding site. Replacement of the engineeredconjunction nucleating molecule with the endogenous conjunctionnucleating molecule reforms the naturally occurring anchorsequence-mediated conjunction.

In some embodiments, a composition or method is described comprising amembrane translocating polypeptide is a targeting moiety. In someembodiments, the membrane translocating polypeptide is a delivery agentthat aids delivery of the targeting moiety described herein.

Irreversible Disruption

In some embodiments, compositions or methods are described herein forirreversibly disrupting an anchor sequence-mediated conjunction. Forexample, the disruption stably modulates transcription forming anon-naturally occurring anchor sequence-mediated conjunction, e.g., amodulation that persists for at least about 1 hr to about 30 days, or atleast about 2 hrs, 6 hrs, 12 hrs, 18 hrs, 24 hrs, 2 days, 3, days, 4days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days,13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days,21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days,29 days, 30 days, or longer or any time therebetween.

In some embodiments, the interaction between a conjunction nucleatingmolecule and the anchor sequence is blocked with a targeting moiety. Inone embodiment, a composition or method is described for disrupting ananchor sequence-mediated conjunction with an epigenetic modifying agent,such as a gene editing system, to target DNA in the vicinity of theanchoring sequence for editing. gRNA recognizes specific DNA sequences(e.g., an anchor sequence, a CTCF anchor sequence, flanked by sequencesthat confer sequence specificity) and RNA-guided nuclease introducesbreaks in the DNA strands, e.g., addition, deletion, homologousrecombination.

In some embodiments, a targeted alteration described herein irreversiblydisrupts the anchor sequence-mediated conjunction. In one embodiment, acomposition or method is described for altering an anchorsequence-mediated conjunction, e.g., by substituting, adding or deletingone or more nucleotides or changing an orientation of at least onecommon nucleotide sequence, with a targeting moiety, e.g., a geneediting system. In one embodiment, a composition or method is describedfor altering one or more DNA methylation sites, e.g., mutatingmethylated cysteine to thymine, with a targeting moiety, e.g., a smallmolecule, e.g., bisulfate compound, within the anchor sequence-mediatedconjunction.

In some embodiments, a targeted alteration described herein irreversiblydisrupts a naturally occurring anchor sequence-mediated conjunction andforms a non-naturally occurring anchor sequence-mediated conjunction. Insome embodiment, a composition or method is described for disrupting ananchor sequence-mediated conjunction with an epigenetic modifying agent,such as a gene editing system, by adding an exogenous anchor sequence toform a non-naturally occurring anchor sequence-mediated conjunction.

In some embodiments, a targeted alteration described herein irreversiblydisrupts an anchor sequence-mediated conjunction. In some embodiment, acomposition or method is described for disrupting an anchorsequence-mediated conjunction with an epigenetic modifying agent, suchas a gene editing system, that elminates a gene for a conjunctionnucleating molecule.

In some embodiments, a targeting moiety that permanently interferes withloop formation by, e.g., CTCF and CTCF-binding motif by blocking theinteraction between CTCF and the CTCF-binding motif. In one embodiment,a composition or method is described for disrupting an anchorsequence-mediated conjunction with an epigenetic modifying agent, anepigenetic modifying agent that covalently binds a conjunctionnucleating molecule sequence to act as a steric blocker. In someembodiments, the epigenetic modifying agent is combined with one or morepeptides, e.g., S-adenosyl methionine (SAM), to act as a steric presenceto interfere with a conjunction nucleating molecule.

Physical Modification

In some embodiments, compositions, agents, fusion molecules, and/ormethods are described for altering an anchor sequence-mediatedconjunction by site-specific disruption at a target anchor sequence. Insome embodiments, such a disruption is achieved using an agent thatphysically interferes with formation and/or maintanence of ananchor-mediated sequence conjunction, e.g., interferes with bindingbetween an anchor sequence and a nucleating agent. In some embodiments,the agent disrupts binding between an anchor sequence and a nucleatingagent via steric inhibition.

In some embodiments, the present disclosure provides a site-specificdisrupting agent, comprising: a DNA-binding moiety (such as aDNA-binding moiety or targeting moiety as described herein) that bindsspecifically to one or more target anchor sequences within a cell andnot to non-targeted anchor sequences within the cell with sufficientaffinity that it competes with binding of an endogenous nucleatingpolypeptide within the cell.

Any of a variety of or combination of the DNA-binding moieties ortargeting moieties as described herein can be used. For example,possible DNA-binding moieties include, but are not limited to, SyntheticNucleic Acids (SNAs), Peptide Nucleic Acids (PNAs), Locked Nucleic Acids(LNAs), Bridged Nucleic Acids (BNAs), polyamide-SNA/LNA/BNA/PNAconjugates, DNA intercalating agents (e.g., SNA/LNA/BNA/PNA conjugates),and DNA sequence-specific binding peptide- or protein-SNA/LNA/PNA/BNAconjugates.

In some embodiments, the site-specific disrupting agent furthercomprises a negative effector moiety (such as any one of or anycombination of negative effector moieties described herein) associatedwith the DNA-binding moiety so that, when the DNA-binding moiety isbound at the one or more target anchor sequences, the negative effectormoiety is localized thereto, the negative effector moiety beingcharacterized in that dimerization of the endogenous nucleatingpolypeptide is reduced when the negative effector moiety is present ascompared with when it is absent.

Genetic Modification

In some embodiments, compositions, agents, fusion molecules, and/ormethods are described for altering an anchor sequence-mediatedconjunction by site specific editing or mutating of an anchor sequenceassociated with a targeted conjunction. An endogenous or naturallyoccurring anchor sequence may be altered to inactivate or delete theanchor sequence (e.g., thereby disrupting an anchor sequence-mediatedconjunction), or may be altered to mutate or replace the anchor sequence(e.g., to mutate or replace an anchor sequence with an altered anchorsequence that has an altered affinity, e.g., decreased affinity orincreased affinity, to a nucleating protein) to modulate the strength ofa targeted conjunction. For example, one or a plurality of exogenousanchor sequences can be incorporated into the genome of a subject tocreate a non-naturally occurring anchor sequence-mediated conjunctionthat incorporates a target gene, e.g., in order to silence the targetgene. In another example, an exogenous anchor sequence can form ananchor sequence-mediated conjunction with an endogenous anchor sequence.The nucleating protein may be, e.g., CTCF, cohesin, USF1, YY1, TAF3,ZNF143 binding motif, or another polypeptide that promotes the formationof an anchor sequence-mediated conjunction.

In one embodiment, a composition or method is described for altering ananchor sequence which is a CTCF-binding motif:N(T/C/G)N(G/A/T)CC(A/T/G)(C/G)(C/T/A)AG(G/A)(G/T)GG(C/A/T)(G/A)(C/G)(C/T/A)(G/A/C)(SEQ ID NO:1), where N is any nucleotide. A CTCF-binding motif may alsobe altered to be in the opposite orientation, e.g.,(G/A/C)(C/T/A)(C/G)(G/A)(C/A/T)GG(G/T)(G/A)GA(C/T/A)(C/G)(A/T/G)CC(G/A/T)N(T/C/G)N(SEQ ID NO:2).

The alteration can be introduced in the gene of a cell, e.g., in vitro,ex vivo, or in vivo.

In some cases, a composition or method is described for altering thechromatin structure, e.g., such that a two-dimensional representation ofthe chromatin structure may change from that of a loop to a non-loop (orfavor a non-loop over a loop) or vice versa, to inactivate the targetedCTCF-binding motif, e.g., the alteration abolishes CTCF binding therebyabolishing the formation of a targeted conjunction. In other examples,the alteration attenuates (e.g., decreases the level of) CTCF binding,thereby decreasing the formation of a targeted conjunction (e.g., byaltering the CTCF sequence to bind with less affinity to a nucleatingprotein). In some embodiments, a targeted alteration increases CTCFbinding by a nucleating protein (e.g., by altering the CTCF sequence tobind with more affinity to a nucleating protein), thereby promoting theformation of a targeted conjunction. The nucleating protein may be,e.g., CTCF, cohesin, USF1, YY1, TAF3, ZNF143 binding motif, or anotherpolypeptide that promotes the formation of an anchor sequence-mediatedconjunction.

As can be appreciated by those of skill in the art, a variety of thecompositions, agents, and/or fusion molecules described herein may besuitable for genetically modifying an anchor sequence, e.g., a targetedanchor sequence.

For example, in some embodiments, provided are fusion moleculescomprising a site-specific targeting moiety (such as any one of thetargeting moieties as described herein) and a deaminating agent, whereinthe site-specific targeting moiety targets the fusion molecule to atarget anchor sequence but not to at least one non-target anchorsequence. A variety of deaminating agents can be used, such asdeaminating agents that do not have enzymatic activity (e.g., chemicalagents such as sodium bisulfate), and/or deaminating agents that haveenzymatic activity (e.g., a deaminase or functional portion thereof).

In some embodiments, provided are pharmaceutical compositions comprisingfusion molecules as described herein.

In some embodiments, provided are compositions (e.g., pharmaceuticalcompositions) comprising (i) a fusion molecule comprising anenzymatically inactive Cas polypeptide and a deaminating agent, or anucleic acid encoding the fusion molecule; and (ii) a guide RNA, whereinthe guide RNA targets the fusion molecule to a target anchor sequencebut not to at least one non-target anchor sequence (a “site-specificguide RNA”, such as described further herein).

For introducing small mutations or a single-point mutation, a homologousrecombination (HR) template can also be used. In one embodiment, the HRtemplate is a single stranded DNA (ssDNA) oligo or a plasmid. For ssDNAoligo design, one may use around 100-150 bp total homology with themutation introduced roughly in the middle, giving 50-75 bp homologyarms. In embodiments, a gRNA for targeting a target anchor sequence,e.g., a CTCF binding motif, is administered in combination with an HRtemplate selected from:

-   -   (a) a nucleotide sequence comprising SEQ ID NO:1;    -   (b) a nucleotide sequence at least 75%, 80%, 85%, 90%, 95%        identical to SEQ ID NO:1;    -   (c) a nucleotide sequence comprising SEQ ID NO:1 having at least        1, 2, 3, 4, 5, but less than 15, 12 or 10 nucleotide additions,        substitutions or deletions.    -   (d) a nucleotide sequence comprising SEQ ID NO:2;    -   (e) a nucleotide sequence at least 75%, 80%, 85%, 90%, 95%        identical to SEQ ID NO:2;    -   (f) a nucleotide sequence comprising SEQ ID NO:2 having at least        1, 2, 3, 4, 5, but less than 15, 12 or 10 nucleotide additions,        substitutions or deletions.

Epigenetic Modification

In some embodiments, compositions and methods are described herein foraltering an anchor sequence-mediated conjunction by site specificepigenetic modification (e.g., methylation or demethylation). Anendogenous or naturally occurring anchor sequence may be altered toincrease its methylation (e.g., thereby decreasing binding of anucleating protein to the anchor sequence and disrupting or preventingan anchor sequence-mediated conjunction), or may be altered to decreaseits methylation (e.g., thereby increasing binding of a nucleatingprotein to the anchor sequence and promoting or increasing the strengthof an anchor sequence-mediated conjunction). The nucleating protein maybe, e.g., CTCF, cohesin, USF1, YY1, TAF3, ZNF143 binding motif, oranother polypeptide that promotes the formation of an anchorsequence-mediated conjunction.

As can be appreciated by those of skill in the art, a variety of thecompositions, agents, and/or fusion molecules described herein may besuitable for epigenetically modifying an anchor sequence, e.g., atargeted anchor sequence.

For example, in some embodiments, provided are fusion moleculescomprising a site-specific targeting moiety (such as any one of thetargeting moieties as described herein) and an epigenetic modifyingagent, wherein the site-specific targeting moiety targets the fusionmolecule to a target anchor sequence but not to at least one non-targetanchor sequence. The epigenetic modifying agent can be any one of or anycombination of epigenetic modifying agents as disclosed herein.

For example, fusions of a catalytically inactive endonuclease e.g., adead Cas9 (dCas9, e.g., D10A; H840A) tethered with all or a portion of(e.g., biologically active portion of) an (one or more) effector domaincreate chimeric proteins that can be guided to specific DNA sites by oneor more RNA sequences (sgRNA) to modulate activity and/or expression ofone or more target nucleic acids sequences (e.g., to methylate ordemethylate a DNA sequence).

In some embodiments, fusion of a dCas9 with all or a portion of one ormore effector domains of an epigenetic modifying agent (such as a DNAmethylase or enzyme with a role in DNA demethylation) creates a chimericprotein that is useful in the methods described herein. Accordingly, insome embodiments, a nucleic acid encoding a dCas9-methylase fusion isadministered to a subject in need thereof in combination with asite-specific gRNA or antisense DNA oligonucleotide that targets thefusion to a conjunction anchor sequence (such as a CTCF binding motif),thereby decreasing the affinity or ability of the anchor sequence tobind a conjunction nucleating protein. In other some embodiments, anucleic acid encoding a dCas9-enzyme fusion is administered to a subjectin need thereof in combination with a site-specific gRNA or antisenseDNA oligonucleotide that targets the fusion to a conjunction anchorsequence (such as a CTCF binding motif), thereby increasing the affinityor ability of the anchor sequence to bind a conjunction nucleatingprotein.

In some embodiments, all or a portion of one or more methylase, orenzyme with a role in DNA demethylation, effector domains are fused withthe inactive nuclease, e.g., dCas9. In other aspects, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more methylase,or enzyme with a role in DNA demethylation, effector domains (all or abiologically active portion) are fused with dCas9. The chimeric proteinsdescribed herein may also comprise a linker, e.g., an amino acid linker.In some aspects, a linker comprises 2 or more amino acids, e.g., one ormore GS sequences. In some aspects, fusion of Cas9 (e.g., dCas9) withtwo or more effector domains (e.g., of a DNA methylase or enzyme with arole in DNA demethylation) comprises one or more interspersed linkers(e.g., GS linkers) between the domains. In some aspects, dCas9 is fusedwith 2-5 effector domains with interspersed linkers.

In embodiments, a composition or method is described comprising a gRNAthat specifically targets a CTCF binding motif associated with anoncogene, a tumor suppressor, or a disease associated with a nucleotiderepeat.

Epigenetic modifying agents useful in the methods and compositionsdescribed herein include agents that affect, e.g., DNA methylation,histone acetylation, and RNA-associated silencing. In embodiments, themethods described herein involve sequence-specific targeting of anepigenetic enzyme (e.g., an enzyme that generates or removes epigeneticmarks, e.g., acetylation and/or methylation). Exemplary epigeneticenzymes that can be targeted to an anchor sequence using the CRISPRmethods described herein include DNA methylases (e.g., DNMT3a, DNMT3b,DNMTL), enzymes with a role in DNA demethylation (e.g., the TET familyenzymes catalyze oxidation of 5-methylcytosine to5-hydroxymethylcytosine and higher oxidative derivatives), histonemethyltransferases, histone deacetylase (e.g., HDAC1, HDAC2, HDAC3),sirtuin 1, 2, 3, 4, 5, 6, or 7, lysine-specific histone demethylase 1(LSD1), histone-lysine-N-methyltransferase (Setdb1), euchromatichistone-lysine N-methyltransferase 2 (G9a), histone-lysineN-methyltransferase (SUV39H1), enhancer of zeste homolog 2 (EZH2), virallysine methyltransferase (vSET), histone methyltransferase (SET2), andprotein-lysine N-methyltransferase (SMYD2). Examples of such epigeneticmodifying agents are described, e.g., in de Groote et al. Nuc. AcidsRes. (2012):1-18.

In embodiments, an epigenetic modifying agent useful herein comprises aconstruct described in Koferle et al. Genome Medicine 7.59 (2015):1-3(e.g., at Table 1), incorporated herein by reference.

Exemplary dCAs9 fusion methods and compositions that are adaptable tothe methods and compositions described herein are known and aredescribed, e.g., in Kearns et al., Functional annotation of nativeenhancers with a Cas9-histone demethylase fusion. Nature Methods 12,401-403 (2015); and McDonald et al., Reprogrammable CRISPR/Cas9-basedsystem for inducing site-specific DNA methylation. Biology Open 2016:doi: 10.1242/bio.019067.

In some embodiments, provided are compositions (e.g., pharmaceuticalcompositions) comprising (i) a fusion polypeptide comprising anenzymatically inactive Cas polypeptide and an epigenetic modifyingagent, or a nucleic acid encoding the fusion polypeptide; and (ii) aguide RNA, wherein the guide RNA targets the fusion molecule to a targetanchor sequence but not to at least one non-target anchor sequence(e.g., a “site-specific guide RNA”, such as those described furtherherein).

New Anchor Sequence-Mediated Conjunction

In some embodiments, compositions, agents, fusion molecules, and/ormethods are described for altering an anchor sequence-mediatedconjunction by generating a new anchor sequence associated with atargeted conjunction.

In some embodiments, provided are engineered site-specific nucleatingagents, comprising: an engineered DNA-binding moiety that bindsspecifically to one or more target sequences within a cell and not tonon-targeted sequences within the cell with sufficient affinity that itcompetes binding of an endogenous nucleating polypeptide within thecell; and a nucleating polypeptide dimerization domain associated withthe engineered DNA-binding moiety so that, so that, when the engineeredDNA-binding moiety is bound at the at least one target sequences, thenucleating polypeptide dimerization domain is localized thereto, andeach at least one targeted sequence is a target anchor sequence. whereinthe at least one or more target anchor sequences is positioned relativeto an anchor sequence to which a nucleating polypeptide binds so that,when the nucleating polypeptide dimerization domain is localized to thetarget anchor sequence, interaction between the nucleating polypeptidedimerization domain and the nucleating polypeptide generates ananchor-sequence-mediated conjunction.

In some embodiments, the target anchor sequence does not comprise a CTCFbinding motif.

Genetic Engineering

In one aspect, the disclosure includes compositions and methodscomprising an engineered cell with a targeted alteration in an anchorsequence-mediated conjunction. In another aspect, the disclosureincludes an engineered nucleic acid sequence comprising an anchorsequence-mediated conjunction with a targeted alteration.

In some embodiments, the targeted alteration comprises a substitution,addition or deletion of one or more nucleotides in at least one anchorsequence, e.g., a conjunction nucleating molecule binding sequence,e.g., a CTCF binding motif. In some embodiments, the targeted alterationcomprises an alteration of one or more DNA methylation sites within theanchor sequence-mediated conjunction.

In some embodiments, the targeted alteration comprises at least oneexogenous anchor sequence. In some embodiments, the targeted alterationalters at least one conjunction nucleating molecule binding site, e.g.altering binding affinity for the conjunction nucleating molecule. Insome embodiments, the targeted alteration changes an orientation of atleast one common nucleotide sequence.

In some embodiments, the targeted alteration forms a non-naturallyoccurring anchor sequence-mediated conjunction, such as anintra-chromosomal loop. In some embodiments, the anchorsequence-mediated conjunction is mediated by a first conjunctionnucleating molecule bound to the first anchor sequence, a secondconjunction nucleating molecule bound to the second anchor sequence, andan association between the first and second conjunction nucleatingmolecules. In one such embodiment, the first or second conjunctionnucleating molecule has a binding affinity for the anchor sequencegreater than or less than a reference value, e.g., binding affinity forthe anchor sequence in the absence of the alteration.

In one aspect, the disclosure includes a pharmaceutical compositioncomprising the engineered cell, e.g., plurality of cells, or theengineered nucleic acid sequence, e.g., a vector, described herein.

In some embodiments, the engineered cell or the engineered nucleic acidsequence described herein comprises a targeted alteration that disruptsthe anchor sequence-mediated conjunction, e.g., reversible orirreversible disruption.

Methods of Use

The methods described herein enable breadth over controlling geneactivity, delivery, and penetrance, e.g., in a cell. In someembodiments, the cell is a mammalian cell. In some embodiments, the cellis a somatic cell. In some embodiments, the cell is a primary cell. Forexample, in some embodiments, the cell is a mammalian somatic cell. Insome embodiments, the mammalian somatic cell is a primary cell. In someembodiments, the mammalian somatic cell is a non-embryonic cell.

In some embodiments, provided are methods comprising a step of:delivering a composition, agent, or fusion molecule to a cell.

In some embodiments, the step of delivering is performed ex vivo. Insome embodiments, methods further comrpise, prior to the step ofdelivering, a step of removing the cell (e.g., a mammalian cell) from asubject. In some embodiments, methods further comprise, after the stepof delivering, a step of (b) administering the cells (e.g., mammaliancells) to a subject.

In some embodiments, the step of delivering comprises administering acomposition comprising the composition, agent, or fusion molecule to asubject. In some embodiments, the subject is has a disease or condition.

In some embodiments, the step of delivering comprises delivery across acell membrane.

In some embodiments, provided are methods comprising a step of (a)substituting, adding, or deleting one or more nucleotides of an anchorsequence within a cell, e.g., a mammalian somatic cell. In someembodiments, the step of substituting, adding, or deleting is performedin vivo. In some embodiments, the step of substituting, adding, ordeleting is performed ex vivo.

In some embodiments, the anchor sequence is a genomic anchor sequence inthat the anchor sequence is located in a genome of the cell.

In some embodiments, provided are methods comprising a step ofdelivering a mammalian somatic cell to a subject having a disease orcondition, wherein one or more nucleotides of an anchor sequence withinthe mammalian somatic cell has been substituted, added, or deleted.

In some embodiments, provided are methods comprising a step of: (a)administering somatic mammalian cells to a subject, wherein the somaticmammalian cells were obtained from the subject, and a composition,agent, or fusion molecule as described herein had been delivered ex vivoto the somatic mammalian cells.

In some embodiments, indications that affect any one of the blood,liver, immune system, neuronal system, etc. or combinations thereof maybe treated by modulating gene expression through altering an anchorsequence-mediated conjunction in a mammalian subject. For example,multiple autoimmune conditions improve when IL-10 mediated tolerizingresponses are elicited. However, recombinant IL-10 therapies have yet tobe efficacious. By altering the anchor sequence-mediated conjunctionassociated with the IL-10 gene, expression of IL-10 may be increased toimprove the autoimmune condition. In another example, IL-6 expressionmay be increased by altering its associated anchor sequence-mediatedconjunction to bring its enhancing sequences in closer proximity to theIL-6 gene.

In one aspect, a method is described for altering gene expression oraltering an anchor sequence-mediated conjunction in a mammalian subject.The method includes administering to the subject (separately or in thesame pharmaceutical composition): a protein comprising a firstpolypeptide domain that comprises a Cas or modified Cas protein and asecond polypeptide domain that comprises a polypeptide having DNAmethyltransferase activity [or associated with demethylation ordeaminase activity], or a nucleic acid encoding a protein comprising afirst polypeptide domain that comprises a Cas or modified Cas proteinand a second polypeptide domain that comprises a polypeptide having DNAmethyltransferase activity [or associated with demethylation ordeaminase activity], and at least one guide RNA (gRNA) that targets ananchor sequence of an anchor sequence-mediated conjunction.

The methods and compositions described herein treat disease by stably ortransiently altering an anchor sequence-mediated conjunction ormodulating transcription of a nucleic acid sequence. In someembodiments, chromatin structure or topology of an anchorsequence-mediated conjunction is altered to result in a stablemodulation of transcription, such as a modulation that persists for atleast about 1 hr to about 30 days, or at least about 2 hrs, 6 hrs, 12hrs, 18 hrs, 24 hrs, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or longer orany time therebetween. In some other embodiments, chromatin structure ortopology of an anchor sequence-mediated conjunction is altered to resultin a transient modulation of transcription, such as a modulation thatpersists for no more than about 30 mins to about 7 days, or no more thanabout 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 19hrs, 20 hrs, 21 hrs, 22 hrs, 24 hrs, 36 hrs, 48 hrs, 60 hrs, 72 hrs, 4days, 5 days, 6 days, 7 days, or any time therebetween.

In one aspect, provided are methods of modifying expression of a targetgene, comprising administering to a cell, tissue or subject acomposition, agent, and/or fusion molecule described herein.

In one aspect, the disclosure includes a method of modifying expressionof a target gene, comprising altering an anchor sequence-mediatedconjunction associated with the target gene, wherein the alterationmodulates transcription of the target gene.

In another embodiment, the methods and compositions described herein toalter an anchor sequence-mediated conjunction may be inducible. The useof an inducible alteration to the anchor sequence-mediated conjunctionprovides a molecular switch capable of turning on the alteration, orturning off the alteration when it is not desired. Examples of systemsused for inducing alterations include, but are not limited to aninducible targeting moiety based on a prokaryotic operon, e.g., the lacoperon, transposon Tn10, tetracycline operon, and the like, and aninducible targeting moiety based on a eukaryotic signaling pathway, e.g.steroid receptor-based expression systems, e.g. the estrogen receptor orprogesterone-based expression system, the metallothionein-basedexpression system, the ecdysone-based expression system. In anotherembodiment, the methods and compositions described herein include aninducible conjunction nucleating molecule or other protein thatinteracts with the anchor sequence-mediated conjunction.

In some embodiments, cells or tissue may be excised from a subject andgene expression, e.g., endogenous or exogenous gene expression, may bealtered ex vivo prior to transplantation of the cells or tissues backinto a subject. Any cell or tissue may be excised and used forretransplantation. Some examples of cells and tissues include, but arenot limited to, stem cells, adipocytes, immune cells, myocytes, bonemarrow derived cells, cells from the kidney capsule, fibroblasts,endothelial cells, and hepatocytes. For example, adipose tissue from apatient may be altered ex vivo to increase energy production and lipidutilization. After the adipose tissue is excised, it may be treated withone or more compositions described herein to upregulate UCP-1 or anyother protein that increases the entropy of energy production pathways,or increases lipolysis, such as prolyl-4-hydroxylase domain 2 (PHD2),lipoprotein lipase (LPL), hormone-sensitive lipase (HSL), and perilipin.The modified adipose cells are returned to the patient and act as“furnaces,” e.g., they uptake lipids from the circulation and use themfor energy production. In another example, an effector can be injectedintramuscularly into a subject to manipulate the GLUT-4 loop andincrease its expression to increase glucose uptake from the circulationinto muscle tissue.

In another embodiment, cells or tissues may be altered with one or morecompositions described herein to produce one or more secreted factors.The cells or tissues are modified to express the desired secretedprotein and transplanted back into the subject. For example, adiposetissue can be modified to express energy utilization or lipolysisproteins to increase energy production. In another example, homing orlocation specific cells may be modified to secrete one or more factorsat a target site once introduced into a subject.

In another embodiment, cells or tissues may be altered with one or morecompositions described herein to produce one or more exogenous Currentdelivery technologies may also have inadvertent effects, e.g., genomewide removal of transcription factors from DNA. In some embodiments, themethod described herein modulates transcription of a gene by deliveringthe composition described herein across a membrane without off-target,e.g., widespread or genome-wide, effects, e.g., removal of transcriptionfactors. In one embodiment, delivering the composition described hereinat doses sufficient to increase penetration of the heterologous moietyacross a membrane does not significantly alter off-targettranscriptional activity, e.g., an increase of less than 50%, 40%, 20%,15%, 10%, 5%, 4%, 3%, 2%, 1%, or any percentage therebetween oftranscriptional activity of one or more off-targets as compared toactivity after delivery of the heterologous moiety alone.

The disclosure also includes a method of delivering the compositiondescribed herein to a subject. In embodiments, the composition isdelivered across a cellular membrane, e.g., a plasma membrane, a nuclearmembrane, an organellar membrane. Current polymeric deliverytechnologies increase endocytic rates in certain cell types, usuallycells that preferentially utilize endocytosis, such as macrophages andcancer cells that rely on calcium influx to trigger endocytosis.Although not bound by any particular theory, the polypeptide describedherein is believed to aid movement of the composition across membranestypically inaccessible by most agents.

In some embodiments, the method described herein comprises delivering acomposition at doses sufficient to increase penetration of theheterologous moiety across a membrane described herein into cells withlow endocytic rates. In some embodiments, the method described hereindoes not significantly increase endocytosis in a target cell. In oneembodiment, delivering the composition described herein at dosessufficient to increase penetration of the heterologous moiety across amembrane does not significantly increase endocytosis, e.g., exhibits anincrease of less than about 50%, 40%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%,or any percentage therebetween of endocytosis as compared to delivery ofthe heterologous moiety alone.

In some embodiments, the method of administering a membranetranslocating polypeptide described herein does not significantlyincrease calcium influx. In one embodiment, the method comprisesdelivering the composition described herein at doses sufficient toincrease penetration of the heterologous moiety across a membrane doesnot significantly increase calcium influx, e.g., an increase of no morethan about 50%, 40%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or anypercentage therebetween of calcium influx as compared to delivery of theheterologous moiety alone. In another embodiment, the method comprisesdelivering the composition described herein at doses sufficient toincrease penetration of the heterologous moiety across a membrane withless compartmentalized calcium movement, e.g., less than about 50%, 40%,20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or any percentage therebetween ofcompartmentalized calcium movement as compared to delivery of theheterologous moiety alone.

In some embodiments, the method of administering a membranetranslocating polypeptide described herein delivers the compositiondescribed herein across a membrane independent of endosomes. In oneembodiment, delivering the composition described herein at dosessufficient to increase penetration of the heterologous moiety across amembrane does not significantly increase endosomal activity, e.g., anincrease of less than 50%, 40%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, orany percentage therebetween of endosomal activity as compared todelivery of the heterologous moiety alone.

In one aspect, the disclosure includes a method of delivering thecomposition, where the composition includes a therapeutic heterologousmoiety, e.g., a drug, and the composition increases intracellulardelivery of the therapeutic as compared to the therapeutic alone. Forexample, the composition comprising a membrane translocating polypeptidedescribed herein described herein can penetrate at least the blood-brainbarrier, the placental membrane separating maternal and fetal blood, andthe blood-testis barrier between the Sertoli cells in the seminiferoustubule and the blood. When the composition of the disclosure includes apolypeptide linked to a therapeutic agent that has poor penetrance orbioavailability, the composition increases penetrance or bioavailabilityof the therapeutic. In another example, the composition includes apolypeptide linked to a heterologous moiety that is an inhibitor of ablood-brain barrier efflux pump, e.g., phenylalanine-arginineβ-naphthylamide (PAβN), verampamil, tricyclic chemosensitizers such asphenothiazines Administration of the composition aids in blood-brainbarrier penetration by selectively inhibiting blood-brain barrier effluxpumps, such as P-glycoprotein and Oat3.

In one aspect, the disclosure includes a method of delivering thecomposition to a target tissue or cell (e.g., CD34+ cells, liver,caudate and putamen nuclei of the telencephalon), where the compositionincludes a targeting heterologous moiety, e.g., a receptor ligand, thattargets the specific tissue or cell and a therapeutic heterologousmoiety. Upon administration, the composition increases targeted deliveryof the therapeutic as compared to the therapeutic alone. When thecomposition of the disclosure is used in combination with an existingtherapeutic that suffers from diffusion or off-target effects, thespecificity of the therapeutic is increased. For example, thecomposition described herein includes a polypeptide linked to achemotherapeutic agent and a ligand moiety that specifically binds areceptor on cancer cells. Administration of the composition increasesspecificity of the chemotherapeutic agent to the cancer cells throughthe ligand-receptor interaction.

In one aspect, the disclosure includes a method of intracellulardelivery of a therapeutic comprising contacting a cell or tissue withthe composition described herein. In one embodiment, the therapeutic isthe heterologous moiety linked to the polypeptide described herein, andthe composition increases intracellular delivery of the therapeutic ascompared to the therapeutic alone.

In one aspect, the disclosure includes a method of inducing cell deathcomprising contacting a cell with the composition described herein. Inone embodiment, the composition comprises a polypeptide linked totopoisomerase inhibitor such as topotecan as described herein and anucleic acid sequence specific for a target cell, such as a viral DNAsequence or a mutation in a gene, etc. The polypeptide translocates intothe nucleus of the cell and specifically binds the viral DNA sequence orthe gene mutation. The topoisomerase inhibitor prevents the DNAreplication machinery from repairing double strand breaks in the genomeand the cell ultimately induces apoptosis. In one embodiment, thecomposition comprises a polypeptide linked to topoisomerase inhibitorsuch as topotecan as described herein and a heterologous moiety thatspecifically binds a necrotic cell marker, such as cyclophilin A (CypA),a cytosolic peptidyl-prolyl cis-trans isomerase released early innecrosis, etc. The polypeptide targets cells in the early stages ofnecrosis by binding the necrotic cell marker and the topoisomeraseinhibitor ultimately induces apoptosis to clear the necrotic cells moreefficiently.

In one aspect, the disclosure includes a method of modulating a membraneprotein by contacting a cell with the composition described herein. Inone embodiment, a membrane protein modulator is the heterologous moietylinked to the polypeptide described herein, and contacting thecomposition with the cell results in membrane protein modulation.

In one aspect, the disclosure includes a method of administering thecomposition described herein to a subject to modulate a membraneprotein, such as an ion channel, a cell surface receptor and a synapticreceptor. In one embodiment, a membrane protein modulator is theheterologous moiety linked to the polypeptide described herein, andadministration of the composition results in membrane proteinmodulation.

In one aspect, the disclosure includes a method of non-parenteraladministration of the composition described herein to a subject toincrease efficacy and decrease toxicity of a parenteral therapeutic. Inone embodiment, a parenteral therapeutic is the heterologous moietylinked to the polypeptide described herein, and administration of thecomposition results in increased efficacy and decreased toxicity of theparenteral therapeutic. In one embodiment, the method includes oraldelivery of the composition. In another embodiment, the parenteraltherapeutic treats a mucosal indication.

In one aspect, the disclosure includes a method of contacting thecomposition described herein with a bacteria or pathogen to decreaseinfectious capacity, toxicity or viability of a bacteria or pathogen.

In one aspect, the disclosure includes a method of inducing apoptosis ina cell harboring a mutation comprising providing the compositiondescribed herein. In one embodiment, the polypeptide described herein islinked to one heterologous moiety that is a nucleic acid thatspecifically binds a mutation sequence in the cell and anotherheterologous moiety that induces apoptosis, such as Fas, Fas ligand,neurotrophin receptor, FADD, BID, TPEN, BAM7, cisplatin, cladribine,puromycin, monensin, sulindac sulfone, triptolide, betulinic acid,bufalin, gambogic acid, apicidin, and other known agents.

In another aspect, a kit is described that includes: (a) a nucleic acidencoding a protein comprising a first polypeptide domain that comprisesa Cas or modified Cas protein and a second polypeptide domain, e.g., apolypeptide having DNA methyltransferase activity or associated withdemethylation or deaminase activity, and (b) at least one guide RNA(gRNA) for targeting the protein to an anchor sequence of a targetanchor sequence-mediated conjunction in a target cell. In someembodiments, the nucleic acid encoding a protein and the gRNA are in thesame vector, e.g., a plasmid, an AAV vector, an AAV9 vector. In anotherembodiment, the nucleic acid encoding a protein and the gRNA are inseparate vectors.

Formulation, Delivery, and Administration

In various embodiments, the pharmaceutical compositions described hereinmay be formulated for delivery to a cell and/or to a subject via anyroute of administration. Modes of administration to a subject mayinclude injection, infusion, inhalation, intranasal, intraocular,topical delivery, intercannular delivery, or ingestion. Injectionincludes, without limitation, intravenous, intramuscular,intra-arterial, intrathecal, intraventricular, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid,intraspinal, intracerebro spinal, and intrasternal injection andinfusion. In some embodiments, administration includes aerosolinhalation, e.g., with nebulization. In some embodiments, administrationis systemic (e.g., oral, rectal, nasal, sublingual, buccal, orparenteral), enteral (e.g., system-wide effect, but delivered throughthe gastrointestinal tract), or local (e.g., local application on theskin, intravitreal injection). In one embodiment, the composition isadministered systemically. In another embodiment, the administration isnon-parenteral and the therapeutic is a parenteral therapeutic.

The compositions may be administered once to the subject or,alternatively, multiple administrations may be performed over a periodof time. For example, two, three, four, five, or more administrationsmay be given to the subject during one treatment or over a period oftime. In some embodiments, six, eight, ten, 12, 15 or 20 or moreadministrations may be given to the subject during one treatment or overa period of time as a treatment regimen.

In some embodiments, administrations may be given as needed, e.g., foras long as symptoms associated with the disease, disorder or conditionpersist. In some embodiments, repeated administrations may be indicatedfor the remainder of the subject's life. Treatment periods may vary andcould be, e.g., one day, two days, three days, one week, two weeks, onemonth, two months, three months, six months, a year, or longer.

In various embodiments, the present disclosure includes pharmaceuticalcompositions described herein with a pharmaceutically acceptableexcipient. Pharmaceutically acceptable excipient includes an excipientthat is useful in preparing a pharmaceutical composition that isgenerally safe, non-toxic, and desirable, and includes excipients thatare acceptable for veterinary use as well as for human pharmaceuticaluse. Such excipients may be solid, liquid, semisolid, or, in the case ofan aerosol composition, gaseous.

The pharmaceutical compositions described herein can also be tableted orprepared in an emulsion or syrup for oral administration.Pharmaceutically acceptable solid or liquid carriers may be added toenhance or stabilize the composition, or to facilitate preparation ofthe composition. Liquid carriers include syrup, peanut oil, olive oil,glycerin, saline, alcohols and water. Solid carriers include starch,lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate orstearic acid, talc, pectin, acacia, agar or gelatin. The carrier mayalso include a sustained release material such as glyceryl monostearateor glyceryl distearate, alone or with a wax.

The pharmaceutical preparations are made following the conventionaltechniques of pharmacy involving milling, mixing, granulation, andcompressing, when necessary, for tablet forms; or milling, mixing andfilling for hard gelatin capsule forms. When a liquid carrier is used,the preparation will be in the form of a syrup, elixir, emulsion or anaqueous or non-aqueous solution or suspension. Such a liquid formulationmay be administered directly per os.

The pharmaceutical compositions according to the disclosure may bedelivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given subject. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration.

Pharmaceutical compositions described herein may be formulates forexample including a carrier, such as a pharmaceutical carrier and/or apolymeric carrier, e.g., a liposome, and delivered by known methods to asubject in need thereof (e.g., a human or non-human agricultural ordomestic animal, e.g., cattle, dog, cat, horse, poultry). Such methodsinclude transfection (e.g., lipid-mediated, cationic polymers, calciumphosphate); electroporation or other methods of membrane disruption(e.g., nucleofection) and viral delivery (e.g., lentivirus, retrovirus,adenovirus, AAV). Methods of delivery are also described, e.g., in Goriet al., Delivery and Specificity of CRISPR/Cas9 Genome EditingTechnologies for Human Gene Therapy. Human Gene Therapy. July 2015,26(7): 443-451. doi:10.1089/hum.2015.074; and Zuris et al. Cationiclipid-mediated delivery of proteins enables efficient protein-basedgenome editing in vitro and in vivo. Nat Biotechnol. 2014 Oct. 30;33(1):73-80.

Liposomes are spherical vesicle structures composed of a uni- ormultilamellar lipid bilayer surrounding internal aqueous compartmentsand a relatively impermeable outer lipophilic phospholipid bilayer.Liposomes may be anionic, neutral or cationic. Liposomes arebiocompatible, nontoxic, can deliver both hydrophilic and lipophilicdrug molecules, protect their cargo from degradation by plasma enzymes,and transport their load across biological membranes and the blood brainbarrier (BBB) (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

Vesicles can be made from several different types of lipids; however,phospholipids are most commonly used to generate liposomes as drugcarriers. Vesicles may comprise without limitation DOTMA, DOTAP, DOTIM,DDAB, alone or together with cholesterol to yield DOTMA and cholesterol,DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol.Methods for preparation of multilamellar vesicle lipids are known in theart (see for example U.S. Pat. No. 6,693,086, the teachings of whichrelating to multilamellar vesicle lipid preparation are incorporatedherein by reference). Although vesicle formation can be spontaneous whena lipid film is mixed with an aqueous solution, it can also be expeditedby applying force in the form of shaking by using a homogenizer,sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro,Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011.doi:10.1155/2011/469679 for review). Extruded lipids can be prepared byextruding through filters of decreasing size, as described in Templetonet al., Nature Biotech, 15:647-652, 1997, the teachings of whichrelating to extruded lipid preparation are incorporated herein byreference.

As described herein, additives may be added to vesicles to modify theirstructure and/or properties. For example, either cholesterol orsphingomyelin may be added to the mixture in order to help stabilize thestructure and to prevent the leakage of the inner cargo. Further,vesicles can be prepared from hydrogenated egg phosphatidylcholine oregg phosphatidylcholine, cholesterol, and dicetyl phosphate. (see, e.g.,Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID469679, 12 pages, 2011. doi:10.1155/2011/469679 for review). Alsovesicles may be surface modified during or after synthesis to includereactive groups complementary to the reactive groups on the carriercells. Such reactive groups include without limitation maleimide groups.As an example, vesicles may be synthesized to include maleimideconjugated phospholipids such as without limitation DSPE-MaL-PEG2000.

A vesicle formulation may be mainly comprised of natural phospholipidsand lipids such as 1,2-distearoryl-sn-glycero-3-phosphatidyl choline(DSPC), sphingomyelin, egg phosphatidylcholines andmonosialoganglioside. Formulations made up of phospholipids only areless stable in plasma. However, manipulation of the lipid membrane withcholesterol reduces rapid release of the encapsulated bioactive compoundinto the plasma or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)increases stability (see, e.g., Spuch and Navarro, Journal of DrugDelivery, vol. 2011, Article ID 469679, 12 pages, 2011.doi:10.1155/2011/469679 for review).

In another embodiment, lipids may be used to form lipid microparticles.Lipids include, but are not limited to, DLin-KC2-DMA4, C12-200 andcolipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG may beformulated (see, e.g., Novobrantseva, Molecular Therapy-Nucleic Acids(2012) 1, e4; doi:10.1038/mtna.2011.3) using a spontaneous vesicleformation procedure. The component molar ratio may be about50/10/38.5/1.5 (DLin-KC2-DMA or C12-200/disteroylphosphatidylcholine/cholesterol/PEG-DMG). Tekmira has a portfolio of approximately95 patent families, in the U.S. and abroad, that are directed to variousaspects of lipid microparticles and lipid microparticles formulations(see, e.g., U.S. Pat. Nos. 7,982,027; 7,799,565; 8,058,069; 8,283,333;7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263; 7,915,399;8,236,943 and 7,838,658 and European Pat. Nos. 1766035; 1519714; 1781593and 1664316), all of which may be used and/or adapted to the presentdisclosure.

Some vesicles and lipid-coated polymer particles are able tospontaneously adsorb to cell surfaces.

The methods and compositions described herein may comprise apharmaceutical composition administered by a regimen sufficient toalleviate a symptom of the disease, disorder or condition. In oneaspect, the disclosure includes a method of delivering a therapeutic byadministering the composition described herein.

Pharmaceutical compositions are also described that include any of thecompositions described herein. In one aspect, a system forpharmaceutical use comprises: a protein comprising a first polypeptidedomain, e.g., a Cas or modified Cas protein, and a second polypeptidedomain, e.g., a polypeptide having DNA methyltransferase activity orassociated with demethylation or deaminase activity, in combination withat least one guide RNA (gRNA) or antisense DNA oligonucleotide thattargets the protein to an anchor sequence of a target anchorsequence-mediated conjunction. The system is effective to alter, in atleast a human cell, the target anchor sequence-mediated conjunction.

In one aspect, a system for pharmaceutical use comprising a compositionthat binds an anchor sequence of an anchor sequence-mediated conjunctionand alters formation of the anchor sequence-mediated conjunction,wherein the composition modulates transcription, in a human cell, of atarget gene associated with the anchor sequence-mediated conjunction.

In one aspect, a system for altering, in a human cell, expression of atarget gene, comprises a targeting moiety (e.g., a gRNA, a membranetranslocating polypeptide) that associates with an anchor sequenceassociated with the target gene, and, optionally, a heterologous moiety(e.g., an enzyme, e.g., a nuclease or deactivated nuclease (e.g., aCas9, dCas9), a methylase, a de-methylase, a deaminase) operably linkedto the targeting moiety, wherein the system is effective to modulate aconjunction mediated by the anchor sequence and alter expression of thetarget gene. The targeting moiety and the heterologous moiety may belinked. In some embodiments, the system comprises a syntheticpolypeptide comprising the targeting moiety and the heterologous moiety.In some embodiments, the system comprises a nucleic acid vector orvectors encoding at least one of the targeting moiety and theheterologous moiety.

In one aspect, a pharmaceutical composition includes a composition thatbinds an anchor sequence of an anchor sequence-mediated conjunction andalters formation of the anchor sequence-mediated conjunction, whereinthe composition modulates transcription, in a human cell, of a targetgene associated with the anchor sequence-mediated conjunction. In someembodiments, the composition disrupts formation of the anchorsequence-mediated conjunction (e.g., decreases affinity of the anchorsequence to a conjunction nucleating molecule, e.g., at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or more). In some embodiments, the composition promotesformation of the anchor sequence-mediated conjunction (e.g., increasesaffinity of the anchor sequence to a conjunction nucleating molecule,e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or more). “Disrupting formation” or“promoting” formation” refers to an alteration in the affinity of theanchor sequence to a conjunction nucleating molecule, e.g., disrupted orpromoted, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.

In some embodiments, the target gene is inside the anchorsequence-mediated conjunction. In some embodiments, the target gene isoutside the anchor sequence-mediated conjunction. In some embodiments,the target gene is inside and outside the anchor sequence-mediatedconjunction. In some embodiments, the composition physically disruptsformation of the anchor sequence-mediated conjunction. For example, thecomposition comprising both targeting and effector activity, e.g.,membrane translocating polypeptide. In some embodiments, the compositioncomprises a targeting moiety (e.g., gRNA, membrane translocatingpolypeptide) that binds the anchor sequence, and is operably linked toan effector moiety that modulates the formation of a conjunctionmediated by the anchor sequence. In some embodiments, the effectormoiety is a chemical, e.g., a chemical that modulates a cytosine (C) oran adenine(A) (e.g., Na bisulfite, ammonium bisulfite). In someembodiments, the effector moiety has enzymatic activity (methyltransferase, demethylase, nuclease (e.g., Cas9), deaminase). In someembodiments, the effector moiety sterically hinders formation of theanchor sequence-mediated conjunction. [e.g., membrane translocatingpolypeptide+nanoparticle].

In another aspect, the disclosure includes a pharmaceutical compositioncomprising (a) a targeting moiety and (b) a DNA sequence comprising ananchor sequence.

In another aspect, the disclosure includes a composition comprising atargeting moiety that binds an anchor sequence of an anchorsequence-mediated conjunction and alters formation of the anchorsequence-mediated conjunction (e.g., alters affinity of the anchorsequence to a conjunction nucleating molecule, e.g., at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or more).

In another aspect, a pharmaceutical composition includes a Cas proteinand at least one guide RNA (gRNA) that targets the Cas protein to ananchor sequence of a target anchor sequence-mediated conjunction. TheCas protein should be effective to cause a mutation of the target anchorsequence that decreases the formation of an anchor sequence-mediatedconjunction associated with the target anchor sequence.

In some embodiments, a gRNA is administered in combination with atargeted nuclease, e.g., a Cas9, e.g., a wild type Cas9, a nickase Cas9(e.g., Cas9 D10A), a dead Cas9 (dCas9), eSpCas9, Cpf1, C2C1, or C2C3, ora nucleic acid encoding such a nuclease. The choice of nuclease andgRNA(s) is determined by whether the targeted mutation is a deletion,substitution, or addition of nucleotides, e.g., a deletion,substitution, or addition of nucleotides to a targeted anchor sequence,e.g., a CTCF binding motif. For example, in some embodiments, one gRNAis administered, e.g., to produce an inactivating indel mutation in ananchor sequence, e.g., a CTCF site, e.g., one gRNA is administered incombination with a nuclease, e.g., wtCas9. As another example, two gRNAsare administered, e.g., in combination with an insertion cassette and anucleic acid encoding a nuclease to produce a replacement sequence atthe targeted anchor sequence. The replacement sequence may have greateror lesser affinity to a nucleating protein, e.g., the replacementsequence may have greater identity to SEQ ID NO:1 or SEQ ID NO:2 thanthe target sequence, e.g., to produce a stronger loop, or lesseridentity to SEQ ID NO:1 or SEQ ID NO:2 than the target sequence, e.g.,to produce a weaker loop. In some embodiments, the replacement sequencehas at least 75%, 80%, 85%, 90%, 95% identity to SEQ ID NO:1 or SEQ IDNO:2. In other embodiments, the replacement sequence has less than 75%,80%, 85%, 90%, 95% identity to SEQ ID NO:1 or SEQ ID NO:2. Thenucleating protein may be, e.g., CTCF, cohesin, USF1, YY1, TAF3, ZNF143binding motif, or another polypeptide that promotes the formation of ananchor sequence-mediated conjunction.

In some embodiments, nucleic acids comprising: a gRNA, a nucleic acidsequence encoding a nuclease, and an insertion cassette are administeredto change the orientation of an anchor sequence, e.g., from being intandem with a partner sequence to being convergent with a partnersequence, e.g., to create a stronger loop, e.g., a gRNA, a nuclease andan insertion cassette are administered to replace an anchor sequencehaving the consensus SEQ ID NO:1 with a sequence having the consensussequence SEQ ID NO:2. In other embodiments, a gRNA, a nucleic acidsequence encoding a nuclease, and an insertion cassette are administeredto change the orientation of an anchor sequence, e.g., from beingconvergent with a partner sequence to being in tandem with a partnersequence, e.g., to create a weaker loop, e.g., a gRNA, a nuclease and aninsertion cassette are administered to replace an anchor sequence havingthe consensus SEQ ID NO:2 with a sequence having the consensus sequenceSEQ ID NO:1.

In one aspect, the disclosure includes a composition comprising anucleic acid or combination of nucleic acids that when administered to asubject in need thereof introduce a site specific alteration (e.g.,insertion, deletion (e.g., knockout), translocation, inversion, singlepoint mutation) in an anchor sequence of an anchor sequence-mediatedconjunction, e.g., a CTCF-binding motif, thereby modulating geneexpression in the subject.

In one aspect, the disclosure includes a pharmaceutical compositioncomprising a guide RNA (gRNA) for use in a clustered regulatoryinterspaced short palindromic repeat (CRISPR) system for gene editing.For example, a gRNA can be administered in combination with a nuclease(e.g., Cpf1 or Cas9) or a nucleic acid encoding the nuclease, tospecifically cleave double-stranded DNA. In the absence of a homologousrepair template, wtCas9 causes non-homologous end joining and results indisrupting the target sequence, e.g., a CTCF binding motif.Alternatively, precise mutations and knock-ins to the target CTCFbinding motif can be made by providing a homologous repair template andexploiting the homology directed repair pathway. Alternatively, doublenicking with paired Cas9 nickases can be used to introduce a staggereddouble-stranded break which can then undergo homology directed repair tointroduce one more nucleotides into the target CTCF binding motif in asite specific manner. Custom gRNA generators and algorithms areavailable commercially for use in developing the methods andcompositions described herein.

In some embodiments, the pharmaceutical composition comprises a zincfinger nuclease (ZFN), or a mRNA encoding a ZFN, that targets (e.g.,cleaves) a CTCF-binding motif.

Methods of Treatment

The compositions and methods described herein can be used to treatdisease in human and non-human animals. In one aspect, the disclosureincludes a method of altering expression of a target gene in a genome,comprising: administering to the genome a pharmaceutical compositioncomprising (a) a targeting moiety and (b) a DNA sequence comprising ananchor sequence, wherein the anchor sequence promotes the formation of aconjunction that brings a gene expression factor (an enhancing sequence,a silencing/repressive sequence) into operable linkage with the targetgene. In one aspect, a method of treating a disease or conditioncomprises administering a targeting moiety selected from at least one ofan exogenous conjunction nucleating molecule, a nucleic acid encodingthe conjunction nucleating molecule, and a fusion of a sequencetargeting polypeptide and a conjunction nucleating molecule to asubject. The table below describes examples of inherited types ofdiseases that can be targeted with the disclosure.

Inheritance Disease Type # of mutated alleles Monoallelic Imprinted 1Hemizygous 1 Autosomal Haploinsufficient 1 Dominant Dominant Negative 1Co-Dominant Biallelic 2 Autosomal Regulatory sequence 2 Recessivemutation ORF mutation 2 Exogenous Viral infection N/A

In some embodiments, the disclosure described herein may also be usefulfor targeting other diseases, e.g., cancer and neurodegeneration. Forexample, oncology indications can be targeted by use of the disclosureto repress oncogenes and/or activate tumor suppressors. Diseasescharacterized by nucleotide repeats, e.g., trinucleotide repeats inwhich silencing of the gene through methylation drives symptoms, can becan be targeted by use of the disclosure to tether the affected gene toan enhancing sequence within an anchor sequence-mediated conjunction.Examples if such diseases include: DRPLA (Dentatorubropallidoluysianatrophy), HD (Huntington's disease), SBMA (Spinal and bulbar muscularatrophy), SCA1 (Spinocerebellar ataxia Type 1), SCA2 (Spinocerebellarataxia Type 2), SCA3 (Spinocerebellar ataxia Type 3 or Machado-Josephdisease), SCA6 (Spinocerebellar ataxia Type 6), SCAT (Spinocerebellarataxia Type 7), SCA17 (Spinocerebellar ataxia Type 17), FRAXA (Fragile Xsyndrome), FXTAS (Fragile X-associated tremor/ataxia syndrome), FRAXE(Fragile XE mental retardation), FRDA (Friedreich's ataxia) FXN or X25,DM (Myotonic dystrophy), SCA8 (Spinocerebellar ataxia Type 8) and SCA12(Spinocerebellar ataxia Type 12). In addition, the genomic loci listedin Table 1 of Herold, et al (Development, 2012) were found to beassociated with CTCF, and diseases related to the genes in the loci maybe targeted by this disclosure as well.

Therapies

The compositions and methods described herein can be used to treatdisease in human and non-human animals. In one aspect, a method oftreating a disease or condition comprises administering the compositiondescribed herein to a subject.

In some embodiments, the subject is a mammal, e.g., a human. In someembodiments, the subject has a disease or condition.

Modulating Gene Expression

In some embodiments, transcription of a nucleic acid sequence ismodulated, e.g., transcription of a target nucleic acid sequence, ascompared with a reference value, e.g., transcription of the targetsequence in the absence of the altered anchor sequence-mediatedconjunction.

In some embodiments, provided are methods of modulating expression of agene associated with an anchor sequence-mediated conjunction, whichconjunction comprises a first anchor sequence and a second anchorsequence. A gene that is associated with an anchor sequence-mediatedconjunction may be at least partially within the conjunction (that is,situated sequence-wise between the first and second anchor sequences),or it may be external to the conjunction in that it is not situatedsequence-wise between the first and second anchor sequences, but islocated on the same chromosome and in sufficient proximity to at leastthe first or the second anchor sequence such that its expression can bemodulated by controlling the topology of the anchor sequence-mediatedconjunction. Those of ordinary skill in the art will understand that thedistance in three-dimensional space between two elements (e.g., betweenthe gene and the anchor sequence-mediated conjunction) may, in someembodiments, be more relevant than the distance in terms of basepairs.In some embodiments, an external but associated gene is located within 2Mb, within 1.9 Mb, within 1.8 Mb, within 1.7 Mb, within 1.6 Mb, within1.5 Mb, within 1.4 Mb, with 1.3 Mb, within 1.3 Mb, within 1.2 Mb, within1.1 Mb, within 1 Mb, within 900 kb, within 800 kb, within 700 kb, within500 kb, within 400 kb, within 300 kb, within 200 kb, within 100 kb,within 50 kb, within 20 kb, within 10 kb, or within 5 kb of the first orsecond anchor sequence.

In some embodiments, modulating expression of the gene comprisesaltering the accessibility of a transcriptional control sequence to thegene. A transcriptional control sequence, whether internal or externalto the anchor sequence-mediated conjunction, can be an enhancingsequence or a silencing (or repressive) sequence.

For example, in some embodiments, provided are methods of modulatingexpression of a gene within an anchor sequence-mediated conjunctioncomprising a step of: contacting the first and/or second anchor sequencewith a composition, agent, and/or fusion molecule as described herein.In some embodiments, the anchor sequence-mediated conjunction comprisesat least one transcriptional control sequence that is “internal” to theconjunction in that it is at least partially located sequence-wisebetween the first and second anchor sequences. Thus, in someembodiments, both the gene whose expression is to be modulated (the“target gene”) and a transcriptional control sequence are within theanchor sequenc-mediated conjunction. See, e.g., a Type 1 anchorsequence-mediated conjunction as depicted in FIG. 6.

In some embodiments, the gene is separated from the internaltranscriptional control sequence by at least 300, at least 400, at least500, at least 600, at least 700, at least 800, or at least 900 basepairs. In some embodiments, the gene is separated from the internaltranscriptional control sequence by at least 1.0, at least 1.2, at least1.4, at least 1.6, or at least 1.8 kb. In some embodiments, the gene isseparated from the internal transcriptional control sequence by at least2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, atleast 7 kb, at least 8 kb, at least 9 kb, or at least 10 kb. In someembodiments, the gene is separated from the internal transcriptionalcontrol sequence by at least 20 kb, at least 30 kb, at least 40 kb, atleast 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90kb, or at least 100 kb. In some embodiments, the gene is separated fromthe internal transcriptional control sequence by at least 150 kb, atleast 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, atleast 400 kb, at least 450 kb, or at least 500 kb. In some embodiments,the gene is separated from the internal transcriptional control sequenceby at least 600 kb, at least 700 kb, at least 800 kb, at least 900 kb,or at least 1 Mb.

In some embodiments, the anchor sequence-mediated conjunction comprisesat least one transcriptional control sequence that is “external” to theconjunction in that it is not located sequence-wise between the firstand second anchor sequences. (See, e.g., Types 2, 3, and 4 anchorsequence-mediated conjunctions depicted in FIG. 6.) In some embodiments,the first and/or the second anchor sequence is located within 1 Mb,within 900 kb, within 800 kb, within 700 kb, within 600 kb, within 500kb, within 450 kb, within 400 kb, within 350 kb, within 300 kb, within250 kb, within 200 kb, within 180 kb, within 160 kb, within 140 kb,within 120 kb, within 100 kb, within 90 kb, within 80 kb, within 70 kb,within 60 kb, within 50 kb, within 40 kb, within 30 kb, within 20 kb, orwithin 10 kb of an external transcriptional control sequence. In someembodiments, the first and/or the second anchor sequence is locatedwithin 9 kb, within 8 kb, within 7 kb, within 6 kb, within 5 kb, within4 kb, within 3 kb, within 2 kb, or within 1 kb of an externaltranscriptional control sequence.

For example, in some embodiments, provided are methods of modulatingexpression of a gene external to an anchor sequence-mediated conjunctioncomprising a step of: contacting the first and/or second anchor sequencewith a composition, agent, and/or fusion molecule as described herein.In some embodiments, the anchor sequence-mediated conjunction comprisesat least one internal transcriptional control sequence.

In some embodiments, the anchor sequence-mediated conjunction comprisesat least one external transcriptional control sequence.

For example, compositions and methods described herein may be used totreat severe congenital neutropenia (SCN). In some embodiments,expression of the Elane gene, which causes the disease, is inhibited. Atargeting moiety is administered to target one or more anchor sequencesadjacent to the Elane gene for alteration and create a repressive loopcomprising the Elane gene.

In one aspect, the disclosure includes a method of treating SCN with apharmaceutical composition described herein. In one embodiment,administration of a composition described herein modulates geneexpression of one or more genes, such as inhibiting gene expression ofthe Elane gene, to treat SCN.

Compositions and methods described herein may be used to treat sicklecell anemia and beta thalassemia. In some embodiments, expression of theHbF from the HBG genes, shown to restore normal hemoglobin levels, isactivated. A targeting moiety is administered to target one or moreanchor sequences adjacent in the HBB gene cluster or the HBG genes. Inone embodiment, an inhibitory loop comprising the HBB gene cluster iscreated. In another embodiment, an activation loop comprising the HBGgenes is created. Downregulating BCL11A has also been shown todownregulate HBB and upregulate HBG expression. In one embodiment, aninhibitory anchor sequence mediated conjunction associated with theBCL11A gene cluster is created.

In one aspect, the disclosure includes a method of treating sickle cellanemia and beta thalassemia with a pharmaceutical composition describedherein. In one embodiment, administration of a composition describedherein modulates gene expression of one or more genes, such asmodulating gene expression from the HBB gene cluster or the HBG genes,to treat SCN.

Compositions and methods described herein may be used to treatMYC-related tumors, e.g., MYC-addicted cancers. In some embodiments,expression of MYC, shown to cause tumors, is inhibited. A targetingmoiety is administered to target one or more anchor sequences adjacentin the MYC gene. In one embodiment, an inhibitory loop comprising theMYC gene is created. In another embodiment, MYC expression is decreasedby disrupting the MYC-associated anchor sequence-mediated conjunction,e.g., decreased transcription due to conformational changes of the DNApreviously open to transcription within the anchor sequence-mediatedconjunction, e.g., decreased transcription due to conformational changesof the DNA creating additional distance between the MYC gene and theenhancing sequence.

In one aspect, the disclosure includes a method of treating MYC-relatedtumors with a pharmaceutical composition described herein. In oneembodiment, administration of a composition described herein modulatesgene expression of one or more genes, such as modulating gene expressionfrom the MYC gene, to treat MYC-related tumors.

The compositions and methods described herein may be used to treatmyoclonic epilepsy of infancy (SMEI or Dravet's syndrome). In someembodiments, loss-of-function mutations in Na_(v)1.1, also known as thesodium channel, voltage-gated, type I, alpha subunit (SCN1A), from theSCN1A gene, cause severe Dravet's syndrome. In one embodiment, atargeting moiety is administered to target one or more anchor sequencesadjacent in the SCN1A gene. In another embodiment, a targeting moiety isadministered to target one or more anchor sequences adjacent in theSCN3A gene to increase expression of Na_(v)1.3, also known as the sodiumchannel, voltage-gated, type III, alpha subunit (SCN3A). In anotherembodiment, a targeting moiety is administered to target one or moreanchor sequences adjacent in the SCN5A gene to increase expression ofNa_(v)1.5, also known as the sodium channel, voltage-gated, type V,alpha subunit (SCN5A). In another embodiment, a targeting moiety isadministered to target one or more anchor sequences adjacent in theSCN8A gene to increase expression of Na_(v)1.6, also known as the sodiumchannel, voltage-gated, type VIII, alpha subunit (SCN8A). In oneembodiment an activation loop comprising any one of SCN1A, SCN3A, SCN5A,and SCN8A genes is created to increase expression of Na_(v)1.1,Na_(v)1.3, Na_(v)1.5, and Na_(v)1.6, respectively.

In one aspect, the disclosure includes a method of treating Dravet'ssyndrome with a pharmaceutical composition described herein. In oneembodiment, administration of a composition described herein modulatesgene expression of one or more genes, such as modulating gene expressionfrom the SCN1A, SCN3A, SCN5A, and SCN8A genes, to treat Dravet'ssyndrome. In another embodiment, administration of a compositioncomprising a membrane translocating polypeptide linked to a GABA agonistto increase GABA activity.

The compositions and methods described herein may be used to treatfamilial erythromelalgia. In some embodiments, loss-of-functionmutations in Na_(v)1.7, also known as the sodium channel, voltage-gated,type IX, alpha subunit (SCN9A), from the SCN9A gene, cause severefamilial erythromelalgia. In one embodiment, a targeting moiety isadministered to target one or more anchor sequences adjacent in theSCN9A gene. In one embodiment an activation loop comprising the SCN9Agene is created to increase expression of Na_(v)1.7.

In one aspect, the disclosure includes a method of treating familialerythromelalgia with a pharmaceutical composition described herein. Inone embodiment, administration of a composition described hereinmodulates gene expression of one or more genes, such as modulating geneexpression from the SCN9A gene, to treat familial erythromelalgia.

The methods described herein may also improve existing therapeutics toincrease bioavailability and/or reduce toxicokinetics.

Bioavailability

In one embodiment, administration of the composition described hereinimproves at least one pharmacokinetic or pharmacodynamic parameter ofthe heterologous moiety, such as targeting, absorption, and transport,as compared to the heterologous moiety alone, or reduces at least onetoxicokinetic parameter, such as diffusion to non-target location,off-target activity, and toxic metabolism, as compared to theheterologous moiety alone (e.g., by at least 5%, 10%, 20%, 25%, 30%,40%, 50%, 60%, 70%, 80% or more). In another embodiment, administrationof the composition described herein increases the therapeutic range ofthe heterologous moiety (e.g., by at least 5%, 10%, 20%, 25%, 30%, 40%,50%, 60%, 70%, 80% or more). In another embodiment, administration ofthe composition described herein reduces the minimum effective dose, ascompared to the heterologous moiety alone (e.g., by at least 5%, 10%,20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or more). In another embodiment,administration of the composition described herein increases the maximumtolerated dose, as compared to the heterologous moiety alone (e.g., byat least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or more). Inanother embodiment, administration of the composition increases efficacyor decreases toxicity of the therapeutic, such as non-parenteraladministration of a parenteral therapeutic. In another embodiment,administration of the composition described herein increases thetherapeutic range of the heterologous moiety while decreasing toxicity,as compared to the heterologous moiety alone (e.g., by at least 5%, 10%,20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or more).

Cancer Therapies

The compositions and methods described herein may be used to treatcancer. The methods described herein may also improve existing cancertherapeutics to increase bioavailability and/or reduce toxicokinetics.Cancer or neoplasm includes solid or liquid cancer and includes benignor malignant tumors, and hyperplasias, including gastrointestinal cancer(such as non-metastatic or metastatic colorectal cancer, pancreaticcancer, gastric cancer, esophageal cancer, hepatocellular cancer,cholangiocellular cancer, oral cancer, lip cancer); urogenital cancer(such as hormone sensitive or hormone refractory prostate cancer, renalcell cancer, bladder cancer, penile cancer); gynecological cancer (suchas ovarian cancer, cervical cancer, endometrial cancer); lung cancer(such as small-cell lung cancer and non-small-cell lung cancer); headand neck cancer (e.g. head and neck squamous cell cancer); CNS cancerincluding malignant glioma, astrocytomas, retinoblastomas and brainmetastases; malignant mesothelioma; non-metastatic or metastatic breastcancer (e.g. hormone refractory metastatic breast cancer); skin cancer(such as malignant melanoma, basal and squamous cell skin cancers,Merkel Cell Carcinoma, lymphoma of the skin, Kaposi Sarcoma); thyroidcancer; bone and soft tissue sarcoma; and hematologic neoplasias (suchas multiple myeloma, acute myelogenous leukemia, chronic myelogenousleukemia, myelodysplastic syndrome, acute lymphoblastic leukemia,Hodgkin's lymphoma).

In one aspect, the disclosure includes a method of treating a cancerwith a pharmaceutical composition described herein. For example, aheterologous moiety of a composition described herein may be ananti-neoplastic agent, chemotherapeutic agent or other anti-cancertherapeutic agent. In one embodiment, administration of a compositiondescribed herein modulates gene expression of one or more genes, such asinhibiting gene expression of an oncogene, to treat the cancer.

For example, oncology indications can be targeted by use of thedisclosure to repress oncogenes (e.g., MYC, RAS, HER1, HER2, JUN, FOS,SRC, RAF, etc.) and/or activate tumor suppressors (e.g., P16, P53, P73,PTEN, RB1, BRCA1, BRCA2, etc.).

In another example, administration of the composition described hereintargets a cancer cell for cell death. The polypeptide is linked to atopoisomerase inhibitor such as topotecan and linked to a nucleic acid,such as through hybridization to the nucleic acid side chains in thepolypeptide. The nucleic acid sequence includes complementary sequencesthat specifically bind the cancer mutation. Upon administration, thepolypeptide translocates into the nucleus to specifically bind thecancer mutation and the topotecan prevents the DNA replication machineryfrom repairing double strand breaks in the genome. The cell ultimatelyinduces apoptosis.

Neurological Diseases or Disorders

The methods described herein may also treat a neurological disease. A“neurological disease” or “neurological disorder” as used herein, is adisease or disorder that affects the nervous system of a subjectincluding a disease that affects the brain, spinal cord, or peripheralnerves. A neurological disease or disorder may affect the nerve cells orthe supporting ells of the nervous system, such as the glial cells. Thecauses of neurological disease or disorder include infection,inflammation, ischemia, injury, tumor, or inherited illness.Neurological diseases or disorders also includes neurodegenerativediseases and myodegenerative diseases. Some examples ofneurodegenerative diseases include, but are not limited to, amyotrophiclateral sclerosis, Alzheimer's disease, frontotemporal dementia,frontotemporal dementia with TDP-43, frontotemporal dementia linked tochromosome-17, Pick's disease, Parkinson's disease, Huntington'sdisease, Huntington's chorea, mild cognitive impairment, Lewy Bodydisease, multiple system atrophy, progressive supranuclear palsy, anα-synucleinopathy, a tauopathy, a pathology associated withintracellular accumulation of TDP-43, and cortico-basal degeneration ina subject. Some other examples of neurological diseases or disordersinclude, but are not limited to, tinnitus, epilepsy, depression, stroke,multiple sclerosis, migraines, and anxiety.

Many bacterial (i.e. Mycobacterial tuberculosis, Neisseriameningitides), viral (i.e. Human Immunodeficiency Virus (HIV),Enteroviruses, West Nile Virus, Zika), fungal (i.e. Cryptococcus,Aspergillus), and parasitic (i.e. malaria, Chagas) infections can affectthe nervous system. Neurological symptoms may occur due to the infectionitself, or due to an immune response.

In one aspect, the disclosure includes a method of treating aneurological disease or disorder with a pharmaceutical compositiondescribed herein. For example, a heterologous moiety of a compositiondescribed herein may be a corticosteroid, an anti-inflammatory, adopamine-affecting drug, or an acetylcholine inhibitor. In oneembodiment, administration of a composition described herein modulatesactivation of a neurotransmitter, neuropeptide, or neuroreceptor.

For example, compositions of the disclosure can be used to modulateneuroreceptor activity (e.g., adrenergic receptor, GABA receptor,acetylcholine receptor, dopamine receptor, serotonin receptor,cannabinoid receptor, cholecystokinin receptor, oxytocin receptor,vasopressin receptor, corticotropin receptor, secretin receptor,somatostatin receptor, etc.) with a neurotransmitter, neuropeptide,agonist or antagonist thereof (e.g., acetylcholine, dopamine,norepinephrine, epinephrine, serotonin, melatonin, cirodhamine,oxytocin, vasopressin, cholecystokinin, neurophysins, neuropeptide Y,enkephalin, orexins, somatostatin, etc.).

Treatments for Acute and Chronic Infections

The methods described herein may also improve existing acute and chronicinfection therapeutics to increase bioavailability and reducetoxicokinetics. As used herein, “acute infection” refers to an infectionthat is characterized by a rapid onset of disease or symptoms. As usedherein, by “persistent infection” or “chronic infection” is meant aninfection in which the infectious agent (e.g., virus, bacterium,parasite, mycoplasm, or fungus) is not cleared or eliminated from theinfected host, even after the induction of an immune response.Persistent infections may be chronic infections, latent infections, orslow infections. While acute infections are relatively brief (lasting afew days to a few weeks) and resolved from the body by the immunesystem, persistent infections may last for months, years, or even alifetime. These infections may also recur frequently over a long periodof time, involving stages of silent and productive infection withoutcell killing or even producing excessive damage to the host cells.Mammals are diagnosed as having a persistent infection according to anystandard method known in the art and described, for example, in U.S.Pat. Nos. 6,368,832, 6,579,854, and 6,808,710.

In some embodiments, the infection is caused by a pathogen from one ofthe following major categories:

i) viruses, including the members of the Retroviridae family such as thelentiviruses (e.g. Human immunodeficiency virus (HIV) anddeltaretroviruses (e.g., human T cell leukemia virus I (HTLV-I), human Tcell leukemia virus II (HTLV-II)); Hepadnaviridae family (e.g. hepatitisB virus (HBV)), Flaviviridae family (e.g. hepatitis C virus (HCV)),Adenoviridae family (e.g. Human Adenovirus), Herpesviridae family (e.g.Human cytomegalovirus (HCMV), Epstein-Barr virus, herpes simplex virus 1(HSV-1), herpes simplex virus 2 (HSV-2), human herpesvirus 6 (HHV-6),varicella-zoster virus), Papillomaviridae family (e.g. HumanPapillomavirus (HPV)), Parvoviridae family (e.g. Parvovirus B19),Polyomaviridae family (e.g. JC virus and BK virus), Paramyxoviridaefamily (e.g. Measles virus), Togaviridae family (e.g. Rubella virus) aswell as other viruses such as hepatitis D virus;

ii) bacteria, such as those from the following families: Salmonella(e.g. S. enterica Typhi), Mycobacterium (e.g. M. tuberculosis and M.leprae), Yersinia (Y. pestis), Neisseria (e.g. N. meningitides, N.gonorrhea), Burkholderia (e.g. B. pseudomallei), Brucella, Chlamydia,Helicobacter, Treponema, Borrelia, Rickettsia, and Pseudomonas;

iii) parasites, such as Leishmania, Toxoplasma, Trypanosoma, Plasmodium,Schistosoma, or Encephalitozoon; and

iv) prions, such as prion protein.

In one embodiment, administration of the composition described hereinsuppresses transcription or activates transcription of one or more genesto treat an infection such as a viral infection. For example, apolypeptide linked to an inhibitor of viral DNA transcription, e.g.,nucleoside analogs such as acyclovir, valaciclovir, penciclovir,denavir, famciclovir, bromovinyldeoxiuridine, ganciclovir; productanalogs such as hydroxycarbamide or pyrophosphate analogs likefoscarnet, allosteric inhibitors or inhibitors that intercalate ordirectly interact with nucleic acids, is administered to treat the viralinfection. The polypeptide may further include a cell targeting ligandfor targeted delivery of the anti-viral therapeutic.

In another example, administration of the composition described hereintargets a virally infected cell for cell death. The polypeptide islinked to a topoisomerase inhibitor such as topotecan and linked to anucleic acid that specifically binds a viral sequence, such as throughhybridization to the nucleic acid side chains in the polypeptide. Thenucleic acid sequence includes complementary sequences that specificallybind viral DNA integrated into the genome. Upon administration, thepolypeptide translocates into the nucleus to specifically bind theintegrated viral DNA and the topotecan prevents the DNA replicationmachinery from repairing double strand breaks in the genome. The cellultimately induces apoptosis.

Treatments of Other Diseases/Disorders/Conditions

Some additional diseases that may be treated by the compositiondescribed herein include, but are not limited to, imprinted orhemizygous mono-allelic diseases, bi-allelic diseases, autosomalrecessive disorders, autosomal dominant disorders, and diseasescharacterized by nucleotide repeats, e.g., trinucleotide repeats inwhich silencing of the gene through methylation drives symptoms, can betargeted by use of the disclosure to modulate expression of the affectedgene. Examples of such diseases include: Jacobsen syndrome, cysticfibrosis, sickle cell anemia, and Tay Sachs disease, tuberous sclerosis,marfan syndrome, neurofibromatosis, retinoblastoma, Waardenburgsyndrome, familial hypercholesterolemia, DRPLA(Dentatorubropallidoluysian atrophy), HD (Huntington's disease),Beckwith-Wiedemann syndrome, Silver-Russell syndrome, SBMA (Spinal andbulbar muscular atrophy), SCA1 (Spinocerebellar ataxia Type 1), SCA2(Spinocerebellar ataxia Type 2), SCA3 (Spinocerebellar ataxia Type 3 orMachado-Joseph disease), SCA6 (Spinocerebellar ataxia Type 6), SCAT(Spinocerebellar ataxia Type 7), SCA17 (Spinocerebellar ataxia Type 17),FRAXA (Fragile X syndrome), FXTAS (Fragile X-associated tremor/ataxiasyndrome), FRAXE (Fragile XE mental retardation), FRDA (Friedreich'sataxia) FXN or X25, DM (Myotonic dystrophy), SCA8 (Spinocerebellarataxia Type 8), and SCA12 (Spinocerebellar ataxia Type 12).

In one aspect, the disclosure includes a method of treating a geneticdisease/disorder/condition with the pharmaceutical composition describedherein. In one embodiment, administration of the composition describedherein modulates gene expression of one or more genes that are indicatedin the genetic disease/disorder/condition, such as activating,suppressing, or modulating expression of the gene.

In one aspect, the disclosure includes a method of treating adisease/disorder/condition with the pharmaceutical composition describedherein. In one embodiment, administration of the composition describedherein modulates gene expression of one or more genes to treat thedisease/disorder/condition, such as activating, suppressing, ormodulating expression of the gene.

All references and publications cited herein are hereby incorporated byreference.

The following examples are provided to further illustrate someembodiments of the present disclosure, but are not intended to limit thescope of the disclosure; it will be understood by their exemplary naturethat other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used.

EXAMPLES

The below Examples demonstrate use of methods, reagents, andcompositions of the present disclosure to modulate expression of a geneassociated with an anchor sequence-mediated conjunction. Unlessdescribed in the past tense, descriptions of experiments are notintended to convey that the experiments have actually been performed.

The present Examples describe, among other things, experiments in cellssuch as cultured cells. However, those of ordinary skill in the artreading the present specification will understand that the presentspecification also teaches application of the disclosed methods, agents,and compositions in a therapeutic context, for example, in mammaliancells that are somatic, non-embryonic, and/or non-cultured (e.g.,primary) (as described further herein).

Example 1: Disruption of a CTCF Anchor Sequence-Mediated Conjunction byGenetic Modification, Epigenetic Modification and Physical Perturbationto Decrease Expression of the MYC Gene

The present Example demonstrates various strategies to decreaseexpression of a gene (in this case, MYC) within a Type 1 anchorsequence-mediated conjunction. Among other things, the present Exampledemonstrates the successful reduction of gene expression by disruptionof the anchor sequence-mediated conjunction via, e.g., modification ofand/or perturbation at a CTCF anchor sequence.

Using methods of the present disclosure that involve using an RNA-guidednuclease domain as part of a targeting moiety, the present Example alsoprovides evidence of synergistic effects when using a combination ofguide RNAs.

MYC (c-Myc) is a regulator gene that encodes a transcription factor thatplays a role in cell cycle progression, apoptosis and cellulartransformation through activation and repression of gene transcription.About 70% of human cancers have been shown to have dysregulation of MYCexpression. MYC inhibition has been explored as a cancer therapeutic anddemonstrated some tumor regression. However, MYC remains difficult totarget intracellularly using conventional pharmacological modalities.

Production of agents: All plasmids and guide RNAs (gRNA) have beenchemically synthesized from commercially available vendors. All agentswere reconstituted in sterile water. All sequences are provided in theMaterials and Methods section.

A1) Genetic Modification by CRISPR/Cas9

This example demonstrates disruption of MYC gene associated CTCF anchorsequence-mediated conjunction by genetic modifications.

A CTCF anchor sequence is located upstream of the MYC gene, allowingenhancers within the loop to influence the MYC promoter. The MYC gene isassociated with an activating enhancer-promoter (E-P) anchorsequence-mediated conjunction.

TABLE 1 Sequences of guide RNAs (gRNAs) targeting putativeCTCF anchor sequences associated with the MYC geneE-P anchor sequence-mediated conjunction. ID Guide RNA Sequence (5′-3′)IHSP-00018 AAAGTAAGTGTGCCCTCTAC

HEK293T cells were transfected with plasmid encoding Cas9 and either co-or serially transfected with a non-targeting gRNA (“Non-targeting,”where the gRNA sequence has no homology to the human genome) or a gRNA,as listed in Table 1, targeted to the CTCF anchor sequence. HEK293Tcells were transfected serially first with a plasmid encoding Cas9, andthen 8 hr later with either a chemically synthesized gRNA targeting theanchor sequence or a non-targeting gRNA (“Non-targeting,” where theguide RNA sequence has no homology to the human genome).

At 72 hr post-transfection, cells were harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific) and genomic DNA was extracted(Qiagen). The resulting cDNA was used for quantitative real-time PCR(Thermo Fisher Scientific).

The locations of potential CTCF binding (black) associated with the MYCgene alongside the locations of the anchor sequences and gRNAs are shownin FIG. 7E.

MYC-specific quantitative PCR probes/primers (Assay ID Hs00153408_m1,Thermo Fisher Scientific) were multiplexed with internal controlquantitative PCR probes/primers, which were either PPIB (Assay IDHs00168719_m1, Thermo Fisher Scientific) or GAPDH (Assay ID Hs02786624g1, Thermo Fisher Scientific) using the FAM-MGB and VIC-MGB dyes,respectively, and gene expression was subsequently analyzed by a realtime PCR kit (Applied Biosystems, Thermo Fisher Scientific). Cellstransfected with guide RNAs proximal to the CTCF anchor sequence showedreduction in MYC expression at 72 hr (FIG. 7A, upper panel). Eachtechnical replicate is represented by empty box symbol.

To detect Cas9-generated genetic modifications (indels), extractedgenomic DNA was used as a template to amplify the anchor sequence DNAregion by PCR (Promega). The resulting PCR products were then subjectedto a nuclease assay (Integrated DNA Technologies) according to themanufacturer's instructions. Cas9-generated indels were detected bysubjecting the resultant PCR products to gel electrophoresis (FIG. 7A,lower panel). Gel electrophoresis images show PCR products of the anchorsequences subjected to the nuclease assay, which cleaves mismatched DNAproducts. The top arrow in the lower panel of FIG. 7A shows theuncleaved PCR products (no Cas9-generated indel) and the bottom arrowshows the cleaved PCR products caused by Cas9-generated indels. Nucleasecleavage products are present in each of the MYC indel Cas9 samples. Theletters A, B and C denote independent biological replicates of eachexperiment. Empty box symbols show technical replicates.

As shown in the FIG. 7A, upper panel, an approximately 40% reduction inMYC gene expression was observed. As shown in the FIG. 7A, upper panel,an approximately 40% reduction in MYC gene expression was observed.

To determine differential CTCF binding at anchor sequences targeted bygRNAs versus non-targeting control gRNAs, a CTCF chromatinimmunoprecipitation-quantitative PCR assay (ChIP-qPCR) is performed. At72 hr post-transfection, HEK293T cells are trypsinized and fixed with 1%formaldehyde in 10% fetal bovine serum and 90% phosphate buffered saline(PBS). Following glycine quenching of fixation, cells are pelleted bycentrifugation, washed and then sonicated using a E220 evolutioninstrument (Covaris) to shear the chromatin. Following anothercentrifugation step, the sheared chromatin supernatant is collected andadded to pre-cleared magnetic beads (Thermo Fisher Scientific) complexedwith a CTCF-specific antibody (Abcam). Following overnight incubation at4° C., the CTCF-chromatin complexes bound to the beads are washed andresuspended in the elution buffer. Subsequently, CTCF-chromatincomplexes are eluted from the beads at 65° C. for 15 min. The crosslinksare then reversed overnight at 65° C., and DNA is purified byphenol:chloroform extraction. The resulting DNA serves as a template forSYBR Green (Thermo Scientific) qPCR using sequence-specific primers(IDT) flanking the CTCF-binding region. The primer sequences used forthe amplification reaction are as follows: 5′-GCTGGAAACCTTGCACCTC-3′ and5′-CGTTCAGGTTTGCGAAAGTA-3′. Diminished input-normalized amplification,by 5% to 100%, indicates reduced CTCF binding due to the targetedgenetic modifications.

A2) Genetic Modification by Cytidine Deaminase-CRISPR/dCas9

This example demonstrates disruption of the MYC gene associated CTCFanchor sequence-mediated conjunction by genome base editing withtargeted cytidine deaminases at and in proximity to the CTCF anchorsequences.

Targeted base editing such as that achieved by a targeted cytidinedeaminase allows genomic editing without creating indels. Withoutwishing to be bound by any particular theory, the inventors proposethat, base editing can provide certain advantages over methods thatinvolve creating indels. For example, base editing may allow moreprecise control over which mutations are induced. Without wishing to bebound by any particular theory, the inventors recognize thatparticularly in therapeutic contexts, increased precision may beparticularly valuable from safety and/or regulatory standpoints.

TABLE 2 Sequences of gRNAs targeting putative CTCF anchorsequences associated with the MYC gene E-P anchorsequence-mediated conjunction. ID Guide RNA Sequence (5′-3′) SACR-00002CTATTCAACCGCATAAGAGA SACR-00011 CGCTGAGCTGCAAACTCAAC SACR-00015GCCTGGATGTCAACGAGGGC SACR-00016 GCGGGTGCTGCCCAGAGAGG SACR-00017GCAAAATCCAGCATAGCGAT

HEK293T cells are transfected with plasmids encoding fusion proteinsconsisting of APOBEC1, a cytidine deaminase that converts cytosine (C)to the RNA base (U), fused to dCas9 and UGI, a uracil glycosylaseinhibitor protein (APOBEC1-dCas9-UGI). Then, 8 hr later, cells aretransfected with chemically synthesized gRNAs tiled at or around theanchor sequence (listed in Table 2), or a non-targeting gRNA(“non-targeting,” where the guide RNA sequence has no homology to thehuman genome).

At 72 hr post-transfection, cells are harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific) and genomic DNA was extracted(Qiagen). The resulting cDNA is used for quantitative real-time PCR(Thermo Fisher Scientific).

For analyzing the conversion of cytosine (C) to uracil (U), gDNAextracted at 72 hr post-transfection (Qiagen) is used as template toamplify the CTCF-binding DNA region by a PCR kit (Promega).APOBEC1-dCas9-UGI-mediated base editing (C→U) is determined bysequencing of the resultant PCR products. By aligning the sequence ofthe resultant PCR products to the original reference sequence of theamplified DNA region, C-to-U editing by APOBEC1-dCas9-UGI is identifiedwhere thymidine (T) is sequenced in place of cytosine (C). Any number ofnon-zero C-to-T sequencing calls on a chromatogram indicate geneticmodification by APOBEC1-dCas9-UGI.

MYC-specific quantitative PCR probes/primers, as described in Example1A.1, are multiplexed with internal control quantitative PCRprobes/primers and gene expression is subsequently analyzed by areal-time PCR kit (Applied Biosystems, Thermo Fisher Scientific). GuideRNAs at or around the CTCF anchor sequence show reduction in MYCexpression after DNA editing by cytidine deamination.

Enzymatic effectors that modify DNA at or near the CTCF anchor sequenceassociated with the MYC gene demonstrate disruption of the MYC geneanchor-mediated conjunction to decrease MYC mRNA levels as compared tothe non-targeting controls.

B) Epigenetic Modification

This example demonstrates disruption of the MYC gene associated CTCFanchor sequence-mediated conjunction by epigenetic modifications.

TABLE 3 Sequences of gRNAs targeting putative CTCF anchorsequences associated with the MYC gene E-P anchorsequence-mediated conjunction. ID Guide RNA Sequence (5′-3′) SACR-00015GCCTGGATGTCAACGAGGGC SACR-00016 GCGGGTGCTGCCCAGAGAGG SACR-00017GCAAAATCCAGCATAGCGAT SACR-00002 CTATTCAACCGCATAAGAGA SACR-00011CGCTGAGCTGCAAACTCAAC SACR-00002 CTATTCAACCGCATAAGAGA SACR-00011CGCTGAGCTGCAAACTCAAC SACR-00017 GCAAAATCCAGCATAGCGAT

HEK293T cells were serially transfected, first with plasmid encodingeither dCas9-DNMT3A-3L (a fusion protein including the active domainsfrom a DNA methyltransferase) or dCas9-KRAB (a transcriptional repressorfusion protein), then 8 hr later with one of five gRNAs tiled around theanchor sequence (listed in Table 3) or a mixture of all five gRNAs tiledaround the anchor sequence (FIG. 7B for dCas9-DNMT3A-3L; FIG. 7C fordCas9-KRAB).

At 72 hr post-transfection, cells were harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific) and genomic DNA was extracted(Qiagen). The resulting cDNA was used for quantitative real-time PCR(Thermo Fisher Scientific).

MYC-specific quantitative PCR probes/primers were multiplexed withinternal control quantitative PCR probes/primers as described in theprevious examples and gene expression was subsequently analyzed by areal time PCR kit (Applied Biosystems, Thermo Fisher Scientific). GuideRNAs proximal to the CTCF anchor sequence showed reduction in MYCexpression at 72 hr after methylation with either dCas9-DNMT3A-3L (FIG.7B) or dCas9-KRAB (FIG. 7C). In FIG. 7B empty boxes are representingdifferent biological replicates. In FIGS. 7C, A and B representbiological replicates while empty boxes denote the value of eachtechnical replicate.

As shown in FIGS. 7A and 7B, transcriptional repression was achievedusing single guides. An approximately 40% or more reduction intranscriptional repression was observed when combinations of gRNAs wereused. As can be seen in FIG. 7B, a synergistic effect was observed witha combination of guide RNAs in that the extent of reduction observedwith the “SACR-00002, 00011, 00015, 00016, 00017” combination wasgreater than the sum of the reductions of observed with each of thegRNAs in the combination.

For analyzing DNA methylation, extracted genomic DNA is bisulfiteconverted using commercially available reagents and protocols (Qiagen),and purified, bisulfite-converted genomic DNA is used as template toamplify the CTCF-binding DNA region by a PCR kit (New England Biolabs).dCas9-DNMT3A-3L-mediated CpG methylation is determined by sequencing theresultant PCR products (bisulfite sequencing). By aligning the sequenceof the resultant PCR products to the unconverted reference DNA sequence,unmethylated CpGs are identified by thymidine (T) base calls where T issequenced in place of C. Thus, CpG methylation is represented by anynumber of non-zero C base calls followed by guanosine (G). The degree ofdCas9-DNMT3A-3L-mediated CpG methylation is subsequently ascertained bycomparing the number and position of C base calls in the MYC-targetedsamples compared to the non-targeting control, where an integer increasein C base calls indicates dCas9-DNMT3A-3L targeted CpG methylation.

Effectors that target epigenetic modifications at or near the CTCFanchor sequence associated with the MYC gene demonstrated disruption ofthe MYC gene anchor-mediated conjunction to decrease MYC mRNA levels ascompared to the non-targeting controls.

Without wishing to be bound by any particular theory, the inventorspropose that, in some embodiments, combinations of gRNAs may be moreeffective that single gRNAs in that such combinations contribute toincreased unwinding of a target nucleic acid, allow improved access toproteins, and/or allow physical displacement of nucleosomes. Theinventors propose that such effects on the target nucleic acid mayreduce steric hindrances and thereby result in enhanced activity ofproteins targeted to the nucleic acid. The inventors propose thatreduction of steric hindrances may be particularly relevant foreffectors such as DNMT3A/3L that act as multimers (e.g., dimers,tetramers, etc.) or are otherwise bulky. The inventors propose that someeffectors such as DNMT3A/3L may act as helicases.

Without wishing to be bound by any particular theory, the inventorspropose that combinations of guide RNAs may, in some embodiments, allowreduction in off-target (non-target) activity (i.e., activity atoff-target sites). The inventors propose that methods in which a robustsynergistic effect can be achieved with multiple guide RNAs (such asmethods involving epigenetic modification, as disclosed herein) areparticularly amenable to fine tuning and/or reduction of off-targetactivity. Such a reduction of off-target activity may improve safety,e.g., in a therapeutic context.

C1) Physical Perturbation with Synthetic Nucleic Acids

This example demonstrates disruption of MYC gene associated CTCF anchorsequence-mediated conjunction by physically preventing CTCF binding atthe anchor sequence.

TABLE 4 Sequences of Synthetic Nucleic Acids (SNAs)targeting putative CTCF anchor sequencesassociated with the MYC gene E-P anchor sequence- mediated conjunction.SNA Sequence (5′-3′) ID (* = phosphothiolate linkage) 5024T*C*C*A*G*GCGCGATGATCTCTGCTGCCAGTAGAGGGCACAC TTACTTTACTTTCG*C*A*A*A*C5025 A*G*G*C*G*CGATGATCTCTGCTGCCAGTAGAGGGCACA*C*T *T*A*C 5026T*G*A*T*C*TCTGCTGCCAGTAGAGGGCACA*C*T*T*A*C 5027G*T*T*T*G*CGAAAGTAAAGTAAGTGTGCCCTCTACTGGCAGC AGAGATCATCGCGC*C*T*G*G*A5028 G*T*A*A*G*TGTGCCCTCTACTGGCAGCAGA*G*A*T*C*A

HEK293T cells are transfected using lipid based transfection reagent(Invitrogen), according to manufacturer's instructions, with SyntheticNucleic Acids (SNAs) located proximally around the CTCF anchor sequencesupstream or downstream of the MYC gene, listed in Table 4, or anon-targeting SNA. At 72 hr post-transfection, cells are harvested forRNA extraction and cDNA is synthesized (Thermo Fisher Scientific)according to the manufacturer's protocols. cDNA is used as a templatefor quantitative real-time PCR.

MYC-specific quantitative PCR probes/primers are multiplexed withinternal control quantitative PCR probes/primers as described in theprevious examples and gene expression is subsequently analyzed by a realtime PCR kit (Applied Biosystems, Thermo Fisher Scientific). Cellstransfected with SNAs proximal to the CTCF anchor sequence are expectedto show reduction in MYC expression.

For determination of differential CTCF binding at anchor sequencestargeted by SNAs versus non-targeting SNAs, a CTCF ChIP-qPCR isperformed on HEK293T cells transfected with various concentrations ofSNAs. The CTCF ChIP protocol is performed as described in Example 1A.1.Phenol:chloroform purified DNA serves as template for SYBR Green (ThermoScientific) qPCR using sequence-specific primers (IDT) flanking theCTCF-binding sequence region. As the SNA dosage increases, acorresponding decrease in the input-normalized amplification of thetarget region demonstrates the displacement of CTCF from anchorsequences due to SNA-targeted physical perturbation.

C2) Physical Perturbation with Targeted Protein Binding

This example demonstrates disruption of MYC gene associated CTCF anchorsequence-mediated conjunction by physically preventing CTCF binding atthe anchor sequence using bulky effector molecules (in this case, fusionproteins).

HEK293T cells were serially transfected, first with plasmid encoding twodifferent dCas9 fusion proteins, then 8 hr later with the gRNA targetedto the CTCF anchor sequence (listed in Table 1).

At 72 hr post-transfection, cells were harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific; Thermo Fisher Scientific) and genomicDNA was extracted (Qiagen). The resulting cDNA was used for quantitativereal-time PCR (Thermo Fisher Scientific).

MYC-specific quantitative PCR probes/primers were multiplexed withinternal control quantitative PCR probes/primers as described in theprevious examples and gene expression was subsequently analyzed by areal time PCR kit (Applied Biosystems, Thermo Fisher Scientific). GuideRNAs proximal to the CTCF anchor sequence showed reduction in MYCexpression at 72 hr after methylation with either dCas9-DNMT3A-3L (FIG.7B) or dCas9-KRAB (FIG. 7C). The letters A and B denote independentbiological replicates of each experiment. Empty box symbols showtechnical replicates.

To determine differential CTCF binding at anchor sequences by targetedgRNAs and protein fusions versus non-targeting control gRNAs and proteinfusions, a CTCF chromatin immunoprecipitation-quantitative PCR assay(ChIP-qPCR) is performed. The CTCF ChIP protocol is performed asdescribed in Example 1A.1. Phenol:chloroform purified DNA serves astemplate for SYBR Green (Thermo Scientific) qPCR using sequence-specificprimers (IDT) flanking the CTCF-binding sequence region. Diminishedinput-normalized amplification indicates reduced CTCF binding due to thetargeted physical disruptions.

Bulky effectors that physically disrupt CTCF binding at CTCF anchorsequences associated with the MYC gene demonstrate disruption of the MYCgene anchor-mediated conjunction and decrease MYC mRNA levels ascompared to the non-targeting controls.

Without wishing to be bound by any particular theory, the inventorspropose that, in some embodiments, bulkiness of effectors may contributeto one or more aspects of disruption. Results shown in FIG. 7B, forexample, were obtained using a bulky fusion protein (dCas9-DNMT3A-3L)that may act as a multimer.

Thus, the present Example demonstrates that methods and agents of thepresent disclosure can be used to substantially reduce expression of agene within a Type 1 loop.

Example 2: Disruption of a YY1 Anchor Sequence-Mediated Conjunction byGenetic Modification, Epigenetic Modification and Physical Perturbationto Decrease Expression of the MYC Gene

The present Example demonstrates various strategies to decreaseexpression of a gene (in this case, MYC) within a Type 1 anchorsequence-mediated conjunction. Among other things, the present Exampledemonstrates the successful reduction of gene expression by disruptionof the anchor sequence-mediated conjunction via, e.g., modification ofand/or perturbation at a YY1 anchor sequence.

The present Example confirms, among other things, that methods andagents of the present disclosure can be applied to modify more than onetype of anchor sequence to modulate expression of a gene associated withan anchor sequence-mediated conjunction.

Additionally, the present Example demonstrates that in the context ofmethods RNA-guided nucleases, substantial changes in gene expression canbe achieved using an individual guide RNA.

Production of agents: All plasmids and guide RNAs have been chemicallysynthesized from commercially available vendors. All agents werereconstituted in sterile water. All sequences are provided in theMaterials and Methods section.

A) Genetic Modification

A YY1 anchor sequence is located upstream of the MYC gene, close towhere distal super-enhancers influence the MYC promoter.

This example demonstrates disruption of MYC gene associated YY1 anchorsequence-mediated conjunction by genetic modifications.

TABLE 5 Sequences of gRNAs targeting putative YY1 anchorsequences associated with the MYCgene E-P anchor sequence-mediated conjunction. IDGuide RNA Sequence (5′-3′) GSSP-00003 TGCAGAAGGTCCGAAGAAAG GSSP-00004AAGAATAACAAGGAGGTGGC

HEK293T cells were serially transfected, first with plasmid encodingCas9 and then 8 hr later with a non-targeting gRNA (“Non-targeting,”where the guide RNA sequence has no homology to the human genome) or agRNA, as listed in Table 5, targeted to the YY1 anchor sequence.

At 72 hr post-transfection, cells were harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific) and genomic DNA was extracted(Qiagen). The resulting cDNA was used for quantitative real-time PCR(Thermo Fisher Scientific).

MYC-specific quantitative PCR probes/primers (Assay ID Hs00153408_m1,Thermo Fisher Scientific) were multiplexed with internal controlquantitative PCR probes/primers, which were PPIB (Assay IDHs00168719_m1, Thermo Fisher Scientific) as described in Example 1 andgene expression was subsequently analyzed by a real time PCR kit(Applied Biosystems, Thermo Fisher Scientific). Guide RNAs proximal tothe YY1 anchor sequence showed reduction in MYC expression at 72 hr(FIG. 7D). Empty box symbols denote the value of each biologicalreplicate.

As shown in the FIG. 7D, substantial reductions of approximately 40% andgreater in MYC gene expression was obtained with individual guides.

To determine differential YY1 binding at anchor sequences by targetedgRNAs versus non-targeting gRNAs, a YY1 chromatinimmunoprecipitation-quantitative PCR assay (ChIP-qPCR) is performed. TheYY1 ChIP protocol is performed as described in Example 1, A1.Phenol:chloroform purified DNA serves as template for SYBR Green (ThermoScientific) qPCR using sequence-specific primers (IDT) flanking theYY1-binding sequence region. Diminished input-normalized amplificationindicates reduced YY1 binding due to the targeted epigeneticmodifications.

Effectors that target epigenetic modifications at or near the YY1 anchorsequence associated with the MYC gene demonstrate disruption of the MYCgene anchor-mediated conjunction to decrease MYC mRNA levels as comparedto the non-targeting controls.

Enzymatic effectors that modify DNA at or near the YY1 anchor sequenceassociated with the MYC gene demonstrated disruption of the MYC geneanchor-mediated conjunction to decrease MYC mRNA levels as compared tothe non-targeting controls.

B) Epigenetic Modification

This example demonstrates disruption of MYC gene associated YY1 anchorsequence-mediated conjunction by epigenetic modifications.

HEK293T cells are serially transfected, first with plasmid encodingeither dCas9-DNMT3A-3L (a fusion protein including the active domainsfrom a DNA methyltransferase) or dCas9-KRAB (a transcriptional repressorfusion protein), then 8 hr later with one of five gRNAs tiled around theanchor sequence (listed in Table 3) or a mixture of all five gRNAs tiledaround the anchor sequence.

At 72 hr post-transfection, cells are harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific; Thermo Fisher Scientific) and genomicDNA is extracted (Qiagen). The resulting cDNA is used for quantitativereal-time PCR (Thermo Fisher Scientific).

MYC-specific quantitative PCR probes/primers are multiplexed withinternal control quantitative PCR probes/primers as described in theprevious examples and gene expression is subsequently analyzed by a realtime PCR kit (Applied Biosystems, Thermo Fisher Scientific). Cellstransfected with guide RNAs proximal to the YY1 anchor sequence areexpected to show reduction in MYC expression after methylation of theYY1 anchor sequences.

To determine differential YY1 binding at anchor sequences by targetedmethyltransferase or transcriptional repressor proteins versusnon-targeted protein fusions, a YY1 chromatinimmunoprecipitation-quantitative PCR assay (ChIP-qPCR) is performed. TheYY1 ChIP protocol is performed as described in Example 1A.1.Phenol:chloroform purified DNA serves as template for SYBR Green (ThermoScientific) qPCR using sequence-specific primers (IDT) flanking theYY1-binding sequence region. Diminished input-normalized amplificationindicates reduced YY1 binding due to the targeted epigeneticmodifications.

Effectors that target epigenetic modifications at or near the YY1 anchorsequence associated with the MYC gene demonstrate disruption of the MYCgene anchor-mediated conjunction and decrease MYC mRNA levels ascompared to the non-targeting controls.

C) Physical Perturbation

This example demonstrates disruption of MYC gene associated YY1 anchorsequence-mediated conjunction by physically preventing YY1 binding atthe anchor sequence.

HEK293T cells are transfected using lipid based transfection reagent(Invitrogen), according to manufacturer's instructions, with SyntheticNucleic Acids (SNAs) listed in Table 4 or a non-targeting SNA. At 72 hrpost-transfection, cells are harvested for RNA extraction and cDNA issynthesized (Thermo Fisher Scientific) according to the manufacturer'sprotocols. cDNA is used as template for quantitative real-time PCR.

MYC-specific quantitative PCR probes/primers are multiplexed withinternal control quantitative PCR probes/primers as described in theprevious examples and gene expression is subsequently analyzed by a realtime PCR kit (Applied Biosystems, Thermo Fisher Scientific). SNAsproximal to the YY1 anchor sequence show reduction in MYC expression.

For determination of differential YY1 binding at anchor sequencestargeted by SNAs versus non-targeting SNAs, a YY1 ChIP-qPCR is performedon HEK293T cells transfected with various concentrations of SNAs. TheYY1 ChIP protocol is performed as described in Example 1A.1.Phenol:chloroform purified DNA serves as template for SYBR Green (ThermoScientific) qPCR using sequence-specific primers (IDT) flanking theYY1-binding sequence region. As the SNA dosage increases, acorresponding decrease in the input-normalized amplification of thetarget region demonstrates the displacement of CTCF from anchorsequences due to SNA-targeted physical perturbation.

Effectors that physically disrupt YY1 binding at YY1 anchor sequencesassociated with the MYC gene demonstrate disruption of the MYC geneanchor-mediated conjunction to decrease MYC mRNA levels as compared tothe non-targeting controls.

Example 3: Disruption of a CTCF Anchor Sequence-Mediated Conjunction byGenetic, Epigenetic, and Physical Perturbation to Decrease Expression ofthe FOXJ3 Gene

The present Example demonstrates various strategies to decreaseexpression of a gene (in this case, FOXJ3) within a Type 1 anchorsequence-mediated conjunction. Among other things, the present Exampledemonstrates the successful site-specific modulation (in this case,repression) of gene expression by disruption of the anchorsequence-mediated conjunction via, e.g., modification of and/orperturbation at a CTCF anchor sequence.

Ovarian cancer is one of the most common cancers and causes of deathamong women in the United States. Forkhead box J3 (FOXJ3) belongs to afamily of transcription factors that plays an important role inregulating the expression of genes involved in cell growth,proliferation, differentiation, and longevity. FOXJ3 has been shown tobe amplified in up to 10% of ovarian cancers, suggesting that disruptionof the FOXJ3 gene anchor-mediated conjunction and decrease in FOXJ3 geneexpression may be therapeutic in ovarian cancer.

The FOXJ3 gene is in a Type 1 anchor sequence-mediated conjunction. Theanchor sequence-mediated conjunction includes the gene encoding FOXJ3and an associated transcription control sequence, e.g., an enhancer.

Production of agents: All plasmids and guide RNAs (gRNA) have beenchemically synthesized from commercially available vendors. All agentswere reconstituted in sterile water. All sequences are provided in theMaterials and Methods section.

A) Genetic Perturbation

This example demonstrates disruption of the FOXJ3 gene Type 1 anchorsequence-mediated conjunction through genetic mutation of the putativeCTCF anchor sequences using CRISPR Cas9.

TABLE 6 Sequences of gRNAs targeting putative CTCF anchorsequences associated with the FOXJ3 gene Type 1anchor sequence-mediated conjunction. ID Guide RNA Sequence (5′-3′)SACR-00055 AGATTCTAAAGGCTGGCTAG SACR-00056 GGGAGCACAGCCCTAAGTAASACR-00057 GAAACCCTCCAAAAGAGGAA SACR-00058 GAGTGCCTGTGGCCACTAGGSACR-00059 GCCTAATTGCAAAGTAGCTT SACR-00060 AGCGACCAGGCGGAGAATGASACR-00061 GGGCCTGAAACAGCACAATG SACR-00062 ACATTGGAGCTGAATGGCCT

HEK293T cells were serially transfected using transfection reagent(Promega), according to the manufacturer's instructions, first withplasmid encoding Cas9, then 8 hr later with chemically synthesized gRNAs(Table 6) that target at or near putative CTCF anchor sequences or anon-targeting gRNA (“Non-targeting,” where the guide RNA sequence has nohomology to the human genome). At 72 hr post-transfection, cells wereharvested for RNA extraction and cDNA was synthesized (Thermo FisherScientific) according to the manufacturer's protocol. The resulting cDNAwas then used for quantitative real-time PCR (Thermo Fisher Scientific).

FOXJ3-specific quantitative PCR probes/primers (Assay ID: Hs00961536,Thermo Fisher Scientific) were multiplexed with internal control PPIBprobes/primers (Assay ID: Hs00168719, Thermo Fisher Scientific) usingthe FAM-MGB and VIC-MGB dyes, respectively, and gene expression wassubsequently analyzed by a real time PCR kit (Applied Biosystems, ThermoFisher Scientific).

The locations of potential CTCF binding (black) associated with theFOXJ3 gene alongside the locations of the anchor sequences (top rightarrows, bottom left arrows) gRNA, and SNAs are shown in FIG. 8A.

The average change of FOXJ3 gene expression in HEK293T cells 72 hrpost-transfection with the indicated gRNAs is shown in FIG. 8B. Eachbiological replicate is depicted by empty box symbols. Guide RNAsproximal to the anchor sequence showed reduction in FOXJ3 mRNA levels.*** p<0.001, ** p<0.01, * p<0.05, n.s. not significant.

As shown in FIG. 8B, a greater decrease in expression was observed withguide RNAs that targeted regions closer to the CTCF binding site.

To determine whether targeting the FOXJ3 anchor sequence-mediatedconjunction does not affect another gene in another anchorsequence-mediated conjunction, HLA-A-specific quantitative PCRprobes/primers (Assay ID: Hs01058806_g1, Thermo Fisher Scientific) weremultiplexed with internal control PPIB probes/primers (Assay ID:Hs00168719, Thermo Fisher Scientific) using the FAM-MGB and VIC-MGBdyes, respectively, and gene expression was subsequently analyzed by areal time PCR kit (Applied Biosystems, Thermo Fisher Scientific).

HLA-A gene expression did not show a significant change in expression inHEK293T cells 72 hr post-transfection with the indicated FOXJ3 gRNAsacross three biological replicates. All of the gRNAs target the anchorsequence of the FOXJ3 anchor sequence-mediated conjunction, and do notshow non-specific effects on HLA-A mRNA levels.

Enzymatic effectors that modify DNA at or near the CTCF anchor sequenceassociated with the FOXJ3 gene demonstrated site-specific disruption ofthe FOXJ3 gene anchor-mediated conjunction and decrease FOXJ3 mRNAlevels as compared to the non-targeting controls.

Thus, the present Example demonstrates modulation at a target genewithout affecting a non-target gene.

B) Epigenetic Perturbation

This example demonstrates disruption of the FOXJ3 gene Type 1 anchorsequence-mediated conjunction by heterochromatin formation at and nearthe anchor sequence. dCas9-KRAB is a transcriptional repressor fusionprotein with enzymatic activity that is specific to the genomic regionsat and in proximity to the anchor sequence, e.g., gRNA binding sites.

TABLE 7 Sequences of gRNAs targeting putative CTCFsites associated with the FOXJ3 geneType 1 anchor sequence-mediated conjunction. IDGuide RNA Sequence (5′-3′) SACR-00064 GACCCTTTGAAGACTCAACT SACR-00065GCTCTGGTAAGGCAAGATTC SACR-00067 AGGTAGCAAATGCCAGCCCA SACR-00069ATCTCTGGATTTCTCATGAG SACR-00071 GCAGTGCTGGGGACAAGATG SACR-00072CTAGGTTAGGTATTGTGCTA SACR-00073 AAGATAAAAGCAGTAGCTAG SACR-00074ATAATAGCAATTAAGAGTAA SACR-00077 TGGAGGCTGCAGGGAGGCGG SACR-00078AATGTGGGCTCCCTCGTCTG

HEK293T cells were serially transfected using transfection reagent(Promega), according to the manufacturer's instructions, first withplasmid encoding dCas9-KRAB, a transcriptional repressor fusion protein,then 8 hr later with mixtures of five chemically synthesized gRNAs,listed in Table 7, located proximally around the anchor sequencesupstream or downstream of FOXJ3 or a non-targeting gRNA(“Non-targeting,” where the guide RNA sequence has no homology to thehuman genome). At 72 hr post-transfection, cells were harvested for RNAextraction and cDNA was synthesized (Thermo Fisher Scientific) accordingto the manufacturer's protocols. cDNA was used as a template forquantitative real-time PCR.

FOXJ3-specific quantitative PCR probes/primers (Assay ID: Hs00961536,Thermo Fisher Scientific) were multiplexed with internal control PPIBquantitative PCR probes/primers (Assay ID: Hs00168719, Thermo FisherScientific) using the FAM-MGB and VIC-MGB dyes, respectively, and geneexpression was analyzed using a real time PCR kit (Applied Biosystems,Thermo Fisher Scientific).

The average change of FOXJ3 gene expression in HEK293T cells 72 hrpost-transfection with the indicated anchor-proximal gRNAs ornon-targeting control gRNA is shown in FIG. 8C. Empty boxes denote thevalue of each biological replicate. Guide RNAs targeting the anchorsequence and flanking sequence regions showed reduction in FOXJ3 mRNAlevels ** p<0.01, * p<0.05.

Effectors that target epigenetic modifications at or near the CTCFanchor sequence associated with the FOXJ3 gene demonstrate disruption ofthe FOXJ3 gene anchor-mediated conjunction and decrease FOXJ3 mRNAlevels as compared to the non-targeting controls.

C) Physical Perturbation

This example demonstrates disruption of the FOXJ3 gene Type 1 anchorsequence-mediated conjunction by physically preventing CTCF binding atthe anchor sequences.

TABLE 8 Sequences of Synthetic Nucleic Acids (SNAs)targeting putative CTCF anchor sequencesassociated with the FOXJ3 gene Type 1anchor sequence-mediated conjunction. SNA Sequence (5′-3′) ID (* =phosphothiolate linkage) 5084 C*C*T*A*G*TGGCCACAGG*C*A*C*T*C 5085G*C*C*C*C*CTAGTGGCCACAGG*C*A*C*T*C 5086 G*A*G*T*G*CCTGTGGCCA*C*T*A*G*G5087 G*A*G*T*G*CCTGTGGCCACTAG*G*G*G*G*C 5088G*T*G*A*G*TGCCTGTGGCCACTAGGGGGCGGGGCTGCCGGC *T*G*T*G*C 5089G*T*G*A*G*TGCCT*G*TGGCCACTAG*G*G*G*GCGG*GGC *T*GCCGGC*T*G*T*G*C 5091A*G*G*G*C*TCCCCGCCAG*C*A*T*G*G 5092 C*C*A*G*C*ATGGTGGCTC*A*C*G*T*C 5093C*C*A*T*G*CTGGCGGGGA*G*C*C*C*T 5094 G*A*C*G*T*GAGCCACCAT*G*C*T*G*G

HEK293T cells were transfected using a lipid based transfection reagent(Invitrogen), according to the manufacturer's instructions, with SNAslocated proximally around the anchor sequences upstream or downstream ofthe FOXJ3 gene, listed in Table 8, or a non-targeting SNA. At 72 hrpost-transfection, cells were harvested for RNA extraction and cDNA wassynthesized (Thermo Fisher Scientific) according to the manufacturer'sprotocols. cDNA was used as template for quantitative real-time PCR.

FOXJ3-specific quantitative PCR probes/primers (Assay ID: Hs00961536,Thermo Fisher Scientific) were multiplexed with internal control PPIBquantitative PCR probes/primers (Assay ID: Hs00168719, Thermo FisherScientific) using the FAM-MGB and VIC-MGB dyes, respectively, and geneexpression was analyzed by a real time PCR kit (Applied Biosystems,Thermo Fisher Scientific).

The average change of FOXJ3 gene expression in HEK293T cells 72 hrpost-transfection with the indicated SNAs is shown in FIG. 8D. Eachbiological replicate is depicted by empty box symbols. SNAs proximal tothe anchor sequence showed reduction in FOXJ3 mRNA levels compared tonon-targeting controls (“Non-targeting,” where the SNA sequence has nohomology to the human genome). This decrease in gene expression issequence specific, as not all target-specific SNAs can modulate FOXJ3mRNA expression. * p<0.05, ** p<0.005, *** p<0.0005. The dose-responsecurve using a non-targeting SNA, and 3 FOXJ3-targeted SNAs with variousdoses for 72 hr post transfection shows a decrease in FOXJ3 mRNA (FIG.8E).

Effectors that physically disrupt CTCF binding at CTCF anchor sequencesassociated with the FOXJ3 gene demonstrate disruption of the FOXJ3 geneanchor-mediated conjunction to decrease FOXJ3 mRNA levels as compared tothe non-targeting controls.

The present Example demonstrates modulation at a target gene usingphysical disruptors. The lack of observed effect on FOXJ3 expressionusing non-targeting controls, and the dose-response curve obtained usingSNAs specific for FOXJ3, support a conclusion that disruption wasachieved in a site-specific manner. Moreover, the observed dose-responseeffect confirms that it is possible to tune the extent of decreased geneexpression using agents and methods of the present disclosure.

Example 4: Disruption of CTCF Anchor Sequence-Mediated Conjunctions byGenetic Modification, Epigenetic Modification and Physical Perturbationto Increase Expression of the TUSC5 Gene

The present Example demonstrates various strategies to increaseexpression of a gene (in this case, TUSC5) within a Type 2 anchorsequence-mediated conjunction. Among other things, the present Exampledemonstrates the successful modulation of gene expression by disruptionof the anchor sequence-mediated conjunction via, e.g., modification ofand/or perturbation at a CTCF anchor sequence.

Tumor suppressor candidate 5 (TUSC5) is a putative transmembraneprotein. TUSC5 is frequently deleted in lung cancers, and is thereforeclassified as a tumor suppressor. Upregulation of TUSC5 might inhibitcancer growth. Thus, upregulation of TUSC5, as demonstrated herein,provided a potential therapeutic strategy. TUSC5 is also highlyexpressed in brown adipose tissue, and potentially involved indifferentiation of brown fat cells.

TUSC5 is located within a CTCF anchor sequence-mediated conjunction. InHEK293T cells, TUSC5 is not expressed, and there are multiple activeenhancers outside this conjunction, both upstream and downstream. Thisconjunction is an example of a Type 2 loop. Disruption of the CTCFanchor sequence at either end of the conjunction is expected to causethe enhancers outside the conjunction to activate expression of TUSC5.

Production of agents: All plasmids and guide RNAs have been chemicallysynthesized from commercially available vendors. All agents werereconstituted in sterile water. All sequences are provided in theMaterials and Methods section.

A) Genetic Modification

This example demonstrates disruption of the TUSC5 gene-associated CTCFanchor sequence-mediated conjunction by genetic modifications.

TABLE 9 Sequences of gRNAs targeting putative CTCFsites associated with the TUSC5 geneType 2 anchor sequence-mediated conjunctions IDGuide RNA Sequence (5′-3′) SACR-00214 CAGCGGATTTGGGCTCCCGG SACR-00216CCTCATCACTACCTGCCACG SACR-00217 CATCACTACCTGCCACGAGG SACR-00218TGAGACTCCAGCATCCCACA SACR-00219 CCAGAGTAGTCCCTGGCACG

HEK293T cells were serially transfected with plasmid encoding Cas9 andeither a non-targeting gRNA (“Non-targeting,” where the guide RNAsequence has no homology to the human genome) or a gRNA, as listed inTable 9, targeted at or near the putative CTCF anchor sequence of theconjunction enclosing the TUSC5 gene. HEK293T cells were transfectedfirst with plasmid encoding Cas9, and then transfected 8 hr later witheither a chemically synthesized gRNAs targeting the anchor sequence or anon-targeting gRNA (“Non-targeting,” where the guide RNA sequence has nohomology to the human genome).

At 72 hr post-transfection, cells were harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific) and genomic DNA was extracted(Qiagen). The resulting cDNA was used for quantitative real-time PCR(Thermo Fisher Scientific).

TUSC5-specific quantitative PCR probes/primers (Assay ID Hs00542659_m1,Thermo Fisher Scientific) were multiplexed with internal controlquantitative PCR probes/primers for PPIB (Assay ID Hs00168719_m1, ThermoFisher Scientific) using the FAM-MGB and VIC-MGB dyes, respectively, andgene expression was subsequently analyzed by a real time PCR kit(Applied Biosystems, Thermo Fisher Scientific).

The average percentage change of TUSC5 gene expression in HEK293T cells72 hr post-transfection with the indicated gRNAs is shown in FIG. 9A.The gRNAs located most proximally to the nucleating agent-binding regionshowed efficacy in upregulating TUSC5 gene expression. Guide RNAsSACR00214 through SACR-00219 showed greater than 5000% increases inTUSC5 mRNA at 72 hr relative to the “Non-targeting” control. Eachbiological replicate is represented by empty box symbol. * p <0.05, **p<0.01, *** p<0.001.

The locations of potential CTCF binding (black) upstream of the TUSC5gene alongside the locations of the gRNAs are shown in FIG. 9B.

Enzymatic effectors that modify DNA at or near the CTCF anchor sequenceassociated with the TUSC5 gene demonstrated disruption of the TUSC5 geneanchor-mediated conjunction and increased TUSC5 mRNA levels as comparedto the non-targeting controls.

To the present inventors' knowledge, the present Example provides thefirst demonstration that an increase of gene expression of thismagnitude (greater than 5000% increase) can be achieved by disrupting ananchor sequence-mediated conjunction with which the gene is associated.

B) Epigenetic Modification

This example demonstrates disruption of the TUSC5 gene associated CTCFanchor sequence-mediated conjunction by epigenetic modifications.

HEK293T cells are serially transfected, first with plasmid encodingeither dCas9-DNMT3A-3L (a fusion protein including the active domainsfrom a DNA methyltransferase) or dCas9-KRAB (a transcriptional repressorfusion protein), then 8 hr later with one of the gRNAs tiled around theanchor sequence (listed in Table 9) or a mixture of gRNAs tiled aroundthe anchor sequence.

At 72 hr post-transfection, cells are harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific) and genomic DNA is extracted(Qiagen). The resulting cDNA is used for quantitative real-time PCR(Thermo Fisher Scientific).

TUSC5-specific quantitative PCR probes/primers are multiplexed withinternal control quantitative PCR probes/primers as described herein andgene expression is subsequently analyzed by a real time PCR kit (AppliedBiosystems, Thermo Fisher Scientific). Cells transfected with guide RNAsproximal to the CTCF anchor sequence are expected to show increases inTUSC5 expression at 72 hr after modification by either dCas9-DNMT3A-3Lor dCas9-KRAB.

Effectors that target epigenetic modifications at or near the CTCFanchor sequence associated with the TUSC5 gene demonstrate disruption ofthe TUSC5 gene anchor-mediated conjunction and increase TUSC5 mRNAlevels as compared to the non-targeting controls.

C) Physical Perturbation

This example demonstrates disruption of TUSC5 gene associated CTCFanchor sequence-mediated conjunction by physically preventing CTCFbinding at the anchor sequence using bulky effector molecules (in thiscase, fusion proteins).

HEK293T cells are serially transfected, first with plasmid encoding twodifferent dCas9 fusion proteins, then 8 hr later with one of the gRNAstiled around the anchor sequence (listed in Table 9) or a mixture ofguide RNAs tiled around the anchor sequence.

At 72 hr post-transfection, cells are harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific; Thermo Fisher Scientific) and genomicDNA is extracted (Qiagen). The resulting cDNA is used for quantitativereal-time PCR (Thermo Fisher Scientific).

TUSC5-specific quantitative PCR probes/primers are multiplexed withinternal control quantitative PCR probes/primers as described in theprevious examples and gene expression is subsequently analyzed by a realtime PCR kit (Applied Biosystems, Thermo Fisher Scientific). Cellstransfected with guide RNAs proximal to the CTCF anchor sequence areexpected to show increases in TUSC5 expression at 72 hr aftermodification by either dCas9-DNMT3A-3L or dCas9-KRAB.

To determine differential CTCF binding at anchor sequences by targetedgRNAs and protein fusions versus non-targeting control gRNAs and proteinfusions, a CTCF chromatin immunoprecipitation-quantitative PCR assay(ChIP-qPCR) is performed. The CTCF ChIP protocol is performed asdescribed in previous examples. Phenol:chloroform purified DNA serves astemplate for SYBR Green (Thermo Scientific) qPCR using sequence-specificprimers (IDT) flanking the CTCF-binding sequence region. Diminishedinput-normalized amplification indicates reduced CTCF binding due to thetargeted physical disruptions.

Bulky effectors that physically disrupt CTCF binding at CTCF anchorsequences associated with the TUSC5 gene demonstrate disruption of theTUSC5 gene anchor-mediated conjunction and increase TUSC5 mRNA levels ascompared to the non-targeting controls.

Example 5: Disruption of a CTCF Anchor Sequence-Mediated Conjunction byGenetic Modification, Epigenetic Modification and Physical Perturbationto Increase Expression of the DAND5 Gene

The present Example demonstrates various strategies to increaseexpression of a gene (in this case, DAND5) within a Type 2 anchorsequence-mediated conjunction. Among other things, the present Exampledemonstrates the successful modulation of gene expression by disruptionof the anchor sequence-mediated conjunction via, e.g., modification ofand/or perturbation at a CTCF anchor sequence.

DAN Domain BMP Antagonist Family Member 5 (DAND5) is a BMP antagonist.DAND5 mutations have been associated with congenital heart defects.

DAND5 is located within a CTCF anchor sequence-mediated conjunction. InHEK293T cells, DAND5 is expressed at very low levels, and there areactive enhancers outside this conjunction upstream of the DAND5 gene.This conjunction is an example of a Type 2 loop. Disruption of the CTCFanchor sequence at the end of the conjunction upstream of DAND5 isexpected to cause the enhancers outside the conjunction to interact withDAND5 and increase its expression.

Production of agents: All plasmids and guide RNAs have been chemicallysynthesized from commercially available vendors. All agents werereconstituted in sterile water. All sequences are provided in theMaterials and Methods section.

A) Genetic Modification

This example demonstrates disruption of the DAND5 gene-associated CTCFanchor sequence-mediated conjunction by genetic modifications.

TABLE 10 Sequences of gRNAs targeting putative CTCFsites associated with the DAND5 geneType 2 anchor sequence-mediated conjunction. IDGuide RNA Sequence (5′-3′) SACR-00187 ACAGCAGAAGGGCAGGTTGG SACR-00188CCAGGACACCCGCCTCCCAG SACR-00189 GCGGCGTGCTCGCCCTCTGG SACR-00190GCATCGCACTCGCAGCTCCG SACR-00191 GGGTGCGAGATAGAGGTGCC SACR-00192GGCACCTCTATCTCGCACCC

HEK293T cells were serially transfected with plasmid encoding Cas9 andeither a non-targeting gRNA (“Non-targeting,” where the gRNA sequencehas no homology to the human genome) or a gRNA, as listed in Table 10,targeted at or near the putative CTCF anchor sequences at end of theconjunction upstream of the DAND5 gene. The HEK293T cells were seriallytransfected first with plasmid encoding Cas9, and then 8 hr later witheither a chemically synthesized gRNAs targeting the CTCF anchor sequenceor a non-targeting gRNA (“Non-targeting,” where the gRNA sequence has nohomology to the human genome).

At 72 hr post-transfection, cells were harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific) and genomic DNA was extracted(Qiagen). The resulting cDNA was used for quantitative real-time PCR(Thermo Fisher Scientific).

DAND5-specific quantitative PCR probes/primers (Assay ID Hs00541488_m1,Thermo Fisher Scientific) were multiplexed with internal controlquantitative PCR probes/primers for PPIB (Assay ID Hs00168719_m1, ThermoFisher Scientific) using the FAM-MGB and VIC-MGB dyes, respectively, andgene expression was subsequently analyzed by a real time PCR kit(Applied Biosystems, Thermo Fisher Scientific). Guide RNA SACR-00189showed a 124% increase in DAND5 expression relative to the“Non-targeting” control, (FIG. 10). Each biological replicate isrepresented by empty box symbol.

The average percentage change of DAND5 gene expression in HEK293T cells72 hr post-transfection with the indicated gRNAs is shown in FIG. 10A.Empty boxes represent each biological replicate. The gRNA closest to thepeak of the CTCF-binding region (SACR-00189) showed efficacy inupregulating DAND5 gene expression. ** p<0.01.

As shown in FIG. 10A, a robust effect (more than 100% increase) on geneexpression was achieved with a single guide RNA targeting the center ofCTCF binding site. In contrast with the results described in Example 4A,no significant increases were observed with guide RNAs targeting regionsnearby, but not at, the middle of the CTCF binding site.

Without wishing to be be bound by any particular theory, the inventorspropose that certain factors (e.g., targeting efficiencies of specificguide RNAs, strength and/or types of nearby transcriptional controlsequences (e.g., enhancers) etc.) may influence a particular locus'ssusceptibility to modulation by disruption of anchor sequence-mediatedconjuctions.

In FIG. 10B, the locations of potential CTCF-binding (black) upstream ofthe DAND5 gene are shown alongside the locations of the gRNAs.

Enzymatic effectors that modify DNA at or near the CTCF anchor sequenceassociated with the DAND5 gene demonstrated disruption of the DAND5 geneanchor-mediated conjunction and increase DAND5 mRNA levels as comparedto the non-targeting controls.

B) Epigenetic Modification

This example demonstrates disruption of the DAND5 gene associated CTCFanchor sequence-mediated conjunction by epigenetic modifications.

HEK293T cells are serially transfected, first with plasmid encodingeither dCas9-DNMT3A-3L (a fusion protein including the active domainsfrom a DNA methyltransferase) or dCas9-KRAB (a transcriptional repressorfusion protein), then 8 hr later with one of the gRNAs tiled around theanchor sequence (listed in Table 10) or a mixture of gRNAs tiled aroundthe anchor sequence.

At 72 hr post-transfection, cells are harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific) and genomic DNA is extracted(Qiagen). The resulting cDNA is used for quantitative real-time PCR(Thermo Fisher Scientific).

DAND5-specific quantitative PCR probes/primers are multiplexed withinternal control quantitative PCR probes/primers as described herein andgene expression is subsequently analyzed by a real time PCR kit (AppliedBiosystems, Thermo Fisher Scientific). Cells transfected with guide RNAsproximal to the CTCF anchor sequence are expected to show increases inDAND5 expression at 72 hr after modification by either dCas9-DNMT3A-3Lor dCas9-KRAB.

Effectors that target epigenetic modifications at or near the CTCFanchor sequence associated with the DAND5 gene demonstrate disruption ofthe DAND5 gene anchor-mediated conjunction and increase DAND5 mRNAlevels as compared to the non-targeting controls.

C) Physical Perturbation

This example demonstrates disruption of DAND5 gene associated CTCFanchor sequence-mediated conjunction by physically preventing CTCFbinding at the anchor sequence using bulky effector molecules (in thiscase, fusion proteins).

HEK293T cells are serially transfected, first with plasmid encoding twodifferent dCas9 fusion proteins, then 8 hr later with one of the gRNAstiled around the anchor sequence (listed in Table 10) or a mixture ofgRNAs tiled around the anchor sequence.

At 72 hr post-transfection, cells are harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific; Thermo Fisher Scientific) and genomicDNA is extracted (Qiagen). The resulting cDNA is used for quantitativereal-time PCR (Thermo Fisher Scientific).

DAND5-specific quantitative PCR probes/primers are multiplexed withinternal control quantitative PCR probes/primers as described in theprevious examples and gene expression is subsequently analyzed by a realtime PCR kit (Applied Biosystems, Thermo Fisher Scientific). Cellstransfected with guide RNAs proximal to the CTCF anchor sequence areexpected to show increases in DAND5 expression at 72 hr aftermodification by either dCas9-DNMT3A-3L or dCas9-KRAB.

To determine differential CTCF binding at anchor sequences by targetedgRNAs and protein fusions versus non-targeting control gRNAs and proteinfusions, a CTCF chromatin immunoprecipitation-quantitative PCR assay(ChIP-qPCR) is performed. The CTCF ChIP protocol is performed asdescribed in previous examples. Phenol:chloroform purified DNA serves astemplate for SYBR Green (Thermo Scientific) qPCR using sequence-specificprimers (IDT) flanking the CTCF-binding sequence region. Diminishedinput-normalized amplification indicates reduced CTCF binding due to thetargeted physical disruptions.

Bulky effectors that physically disrupt CTCF binding at CTCF anchorsequences associated with the DAND5 gene demonstrate disruption of theDAND5 gene anchor-mediated conjunction and increase DAND5 mRNA levels ascompared to the non-targeting controls.

Example 6: Disruption of a CTCF Anchor Sequence-Mediated Conjunction byGenetic Modification, Epigenetic Modification and Physical Perturbationto Decrease Expression of the SHMT2 Gene

The present Example demonstrates various strategies to decreaseexpression of a gene (in this case, SHMT2) within a Type 3 anchorsequence-mediated conjunction. Among other things, the present Exampledemonstrates the successful modulation of gene expression by disruptionof the anchor sequence-mediated conjunction via, e.g., modification ofand/or perturbation at a CTCF anchor sequence.

Serine hydroxymethyltransferase (SHMT2) is a mitochondrial protein thatis involved in the glycine synthesis pathway. SHMT2 is highly expressedin cancer cells in glioblastomas and confers these cells with a survivaladvantage by reducing the requirement for oxygen. SHMT2 might be apotential oncology target.

SHMT2 is located within a CTCF anchor sequence-mediated conjunction. Thenucleosomes in the flanking regions of this conjunction are marked withthe repressive chromatin mark H3KK27me3. This conjunction is thereforean example of a Type 3 loop. Disruption of the CTCF anchor sequence ateither end of the conjunction is expected to cause the spread of theflanking repressive chromatin marks to the SHMT2 gene, thereby causingits downregulation.

Production of agents: All plasmids and guide RNAs have been chemicallysynthesized from commercially available vendors. All agents werereconstituted in sterile water. All sequences are provided in theMaterials and Methods section.

A) Genetic Modification

This example demonstrates disruption of the SHMT2 gene-associated CTCFanchor sequence-mediated conjunction by genetic modifications.

TABLE 11 Sequences of gRNAs targeting putativeCTCF sites associated with the SHMT2 geneType 3 anchor sequence-mediated conjunction IDGuide RNA Sequence (5′-3′) SACR-00149 TGGGCTCGGGCGCCCCCTGG SACR-00151AGGGTCGACACTGCCCGACA SACR-00156 CGGGGCAGGTCTCCCTCTGG SACR-00165CCAGGCGTACAGACACCACC

HEK293T cells were serially transfected with plasmid encoding Cas9 andeither a non-targeting gRNA (“Non-targeting,” where the gRNA sequencehas no homology to the human genome) or a gRNA, as listed in Table 11,targeted at or near the putative CTCF anchor sequences at either end ofthe conjunction enclosing the SHMT2 gene. HEK293T cells were seriallytransfected first with plasmid encoding Cas9, and then 8 hr later witheither a chemically synthesized gRNAs targeting the anchor sequence or anon-targeting gRNA (“Non-targeting,” where the gRNA sequence has nohomology to the human genome).

At 72 hr post-transfection, cells were harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific) and genomic DNA was extracted(Qiagen). The resulting cDNA was used for quantitative real-time PCR(Thermo Fisher Scientific).

SHMT2-specific quantitative PCR probes/primers (Assay ID Hs01059263_g1,Thermo Fisher Scientific) were multiplexed with internal controlquantitative PCR probes/primers for PPIB (Assay ID Hs00168719_m1, ThermoFisher Scientific) using the FAM-MGB and VIC-MGB dyes, respectively, andgene expression was subsequently analyzed by a real time PCR kit(Applied Biosystems, Thermo Fisher Scientific). Cells transfected withguide RNAs SACR-00149 and SACR-00156 showed a 24% and 17% reduction inSHMT2 expression respectively at 72 hr relative to the “Non-targeting”control, while cells transfected with SACR-00151 and SACR-00165 did not(FIG. 11A). Each biological replicate is represented by empty boxsymbol.

The average percentage change of SHMT2 gene expression in HEK293T cells72 h post-transfection with the indicated gRNAs is shown in FIG. 11A.Empty boxes denote the value of each biological replicate. Guide RNAsoverlapping strong CTCF anchor sequences showed effectiveness indownregulating SHMT2 gene expression.

The locations of potential CTCF-binding (black) upstream (FIG. 11B) anddownstream (FIG. 11C) of the SHMT2 gene are shown, alongside thelocations of the gRNAs.

Enzymatic effectors that modify DNA at or near the CTCF anchor sequenceassociated with the SHMT2 gene demonstrated disruption of the DAND5 geneanchor-mediated conjunction to decrease SHMT2 mRNA levels as compared tothe non-targeting controls.

Thus, the present Example demonstrates that modulation of geneexpression can be achieved by disrupting anchor sequences at either endof an anchor sequence-mediated conjunction.

B) Epigenetic Modification

This example demonstrates disruption of the SHMT2 gene associated CTCFanchor sequence-mediated conjunction by epigenetic modification.

TABLE 12 Sequences of gRNAs targeting putative CTCFanchor sequences associated with the SHMT2gene Type 3 anchor sequence-mediated conjunction ID SetGuide RNA Sequence (5′-3′) SACR-00146 Set 1 GCTTGGAGTCCAGTCCCAGCSACR-00148 Set 1 TCAAAGGCAGCGGGACTCAG SACR-00150 Set 1AAGCTCGGGGAAGAGGCCTT SACR-00152 Set 1 CACTCCAGGCACCAACTTAG SACR-00154Set 1 ACTCCCGCCTCCAAGACAGT SACR-00155 Set 2 AAAGAAAGAAAAAAAGCCGCSACR-00157 Set 2 GGGCACAGTAAGATGGAGAG SACR-00162 Set 2GCAGGGGAGGATCTCAGAGT SACR-00164 Set 2 TGGGACACAGACCTCCTACT SACR-00167Set 2 CAGGTGCATAATGAGTGCTG

HEK293T cells were serially transfected, first with plasmid encodingdCas9-KRAB (a transcriptional repressor fusion protein), then 8 hr laterwith two different mixtures (Set 1, Set 2) of five gRNAs (listed inTable 12) tiled around the CTCF anchor sequence (FIGS. 11B and 11C).

At 72 hr post-transfection, cells were harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific) and genomic DNA was extracted(Qiagen). The resulting cDNA was used for quantitative real-time PCR(Thermo Fisher Scientific).

SHMT2-specific quantitative PCR probes/primers were multiplexed withinternal control quantitative PCR probes/primers as described in theprevious examples and gene expression was subsequently analyzed by areal time PCR kit (Applied Biosystems, Thermo Fisher Scientific). Cellstransfected with either of two sets of 5 gRNAs showed reduction in SHMT2expression (Set1: 18%, Set 2: 13%) at 72 hr after repression withdCas9-KRAB (FIG. 11D). Empty boxes denote the value of each biologicalreplicate.

The average percentage change of SHMT2 gene expression in HEK293T cells72 hr post-transfection with the indicated gRNAs is shown in FIG. 11D.Cells transfected with guide RNAs proximal to the strong CTCF anchorsequence showed decreases in SHMT2 expression at 72 hr after treatmentwith dCas9-KRAB. Empty boxes denote the value of each biologicalreplicate. ** p<0.01

Effectors that target epigenetic modifications at or near the CTCFanchor sequence associated with the SHMT2 gene demonstrate disruption ofthe SHMT2 gene anchor-mediated conjunction to decrease SHMT2 mRNA levelsas compared to the non-targeting controls.

C) Physical Perturbation

This example demonstrates disruption of SHMT2 gene associated CTCFanchor sequence-mediated conjunction by physically preventing CTCFbinding at the anchor sequence.

HEK293T cells are serially transfected, first with plasmid encoding twodifferent dCas9 fusion proteins, then 8 hr later with one of the gRNAstiled around the anchor sequence (listed in Table 12) or a mixture ofgRNAs tiled around the anchor sequence.

At 72 hr post-transfection, cells are harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific; Thermo Fisher Scientific) and genomicDNA is extracted (Qiagen). The resulting cDNA is used for quantitativereal-time PCR (Thermo Fisher Scientific).

SHMT2-specific quantitative PCR probes/primers are multiplexed withinternal control quantitative PCR probes/primers as described in theprevious examples and gene expression is subsequently analyzed by a realtime PCR kit (Applied Biosystems, Thermo Fisher Scientific). Cellstransfected with guide RNAs proximal to the CTCF anchor sequence areexpected to showdecreases in SHMT2 expression at 72 hr after treatmentwith dCas9-KRAB.

To determine differential CTCF binding at anchor sequences by targetedgRNAs and protein fusions versus non-targeting control gRNAs and proteinfusions, a CTCF chromatin immunoprecipitation-quantitative PCR assay(ChIP-qPCR) is performed. The CTCF ChIP protocol is performed asdescribed in previous examples. Phenol:chloroform purified DNA serves astemplate for SYBR Green (Thermo Scientific) qPCR using sequence-specificprimers (IDT) flanking the CTCF-binding sequence region. Diminishedinput-normalized amplification indicates reduced CTCF binding due to thetargeted physical disruptions.

Bulky effectors that physically disrupt CTCF binding at CTCF anchorsequences associated with the SHMT2 gene demonstrate disruption of theSHMT2 gene anchor-mediated conjunction and decrease SHMT2 mRNA levels ascompared to the non-targeting controls.

Example 7: Disruption of a CTCF Anchor Sequence-Mediated Conjunction byGenetic Modification to Increase Expression of the TTC21B Gene

The present Example demonstrates various strategies to increaseexpression of a gene (in this case, TTC21B) just outside a Type 2 anchorsequence-mediated conjunction (which contains an enhancer). Among otherthings, the present Example demonstrates the successful modulation ofgene expression by disruption of the anchor sequence-mediatedconjunction via, e.g., modification of and/or perturbation at a CTCFanchor sequence.

Tetratricopeptide repeat domain-containing protein 21B (TTC21B) is anaxonemal protein involved in ciliary function. Hypomorphic alleles ofTTC21B have been associated with human ciliopathies such asnephronophthisis and upregulation of this gene might attenuate theseverity of the disease.

TTC21B is located just outside a CTCF anchor sequence-mediatedconjunction. In HEK293T cells, TTC21B is not expressed, and there is anactive enhancer within the neighboring conjunction. This configurationis an example of a Type 2 loop. Disruption of the CTCF anchorsequence-mediated conjunction is expected to cause the enhancer insidethe conjunction to activate the expression of TTC21B.

Production of agents: All plasmids and guide RNAs have been chemicallysynthesized from commercially available vendors. All agents werereconstituted in sterile water. All sequences are provided in theMaterials and Methods section.

A) Genetic Modification

This example demonstrates disruption of a CTCF anchor sequence-mediatedconjunction by genetic modifications to increase TTC21B gene expression.

TABLE 13 Sequences of gRNAs targeting putative CTCF anchorsequences associated with the TTC21B gene Type2 anchor sequence-mediated conjunction. ID Guide RNA Sequence (5′-3′)SACR-00023 GTTGTTTTACGGCCACAAGG SACR-00024 TTTTTTTCTGCGCCACCTTG

HEK293T cells were transfected with plasmid encoding Cas9 and either co-or serially transfected with a non-targeting gRNA (“Non-targeting,”where the guide RNA sequence has no homology to the human genome) or agRNA, as listed in Table 13, targeted at or near the putative CTCFanchor sequences at either end of the conjunction enclosing the TTC21Bgene. HEK293T cells were serially transfected first with plasmidencoding Cas9, and then 8 hr later with either a chemically synthesizedgRNAs targeting the anchor sequence or a non-targeting gRNA(“Non-targeting,” where the gRNA sequence has no homology to the humangenome).

At 72 hr and 14 days post-transfection, cells were harvested for RNAextraction and cDNA synthesis using commercially available reagents andprotocols (Qiagen; Thermo Fisher Scientific) and genomic DNA wasextracted (Qiagen). The resulting cDNA was used for quantitativereal-time PCR (Thermo Fisher Scientific).

TTC21B-specific quantitative PCR probes/primers (Assay ID Hs01095195_m1,Thermo Fisher Scientific) were multiplexed with internal controlquantitative PCR probes/primers for PPIB (Assay ID Hs00168719_m1, ThermoFisher Scientific) using the FAM-MGB and VIC-MGB dyes, respectively, andgene expression was subsequently analyzed by a real time PCR kit(Applied Biosystems, Thermo Fisher Scientific). Cells transfected withgRNA SACR-00024 showed a trend of upregulating TTC21B expression after72 hours (FIG. 12A). After 14 days, cells transfected with gRNASACR-00024 showed a 29% increase in TTC21B expression relative to the“Non-targeting” control, (FIG. 12B). Empty boxes denote the value ofeach biological replicate.

Enzymatic effectors that modify DNA at or near the CTCF anchor sequencedemonstrated disruption of the anchor-mediated conjunction and increasedTTC21B mRNA levels as compared to the non-targeting controls.

B) Epigenetic Modification

This example demonstrates disruption of the CTCF anchorsequence-mediated conjunction by epigenetic modifications to increaseTTC21B gene expression.

HEK293T cells are serially transfected, first with plasmid encodingeither dCas9-DNMT3A-3L (a fusion protein including the active domainsfrom a DNA methyltransferase) or dCas9-KRAB (a transcriptional repressorfusion protein), then 8 hr later with one of the gRNAs tiled around theanchor sequence (listed in Table 13) or a mixture of both gRNAs.

At 14 days post-transfection, cells are harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific) and genomic DNA is extracted(Qiagen). The resulting cDNA is used for quantitative real-time PCR(Thermo Fisher Scientific).

TTC21B-specific quantitative PCR probes/primers are multiplexed withinternal control quantitative PCR probes/primers as described herein andgene expression is subsequently analyzed by a real time PCR kit (AppliedBiosystems, Thermo Fisher Scientific). Cells transfected with guide RNAsproximal to the CTCF anchor sequence are expected to show increases inTTC21B expression at 14 days after modification by eitherdCas9-DNMT3A-3L or dCas9-KRAB.

Effectors that target epigenetic modifications at or near the CTCFanchor sequence adjacent to the TTC21B gene demonstrate disruption ofthe gene anchor-mediated conjunction and increase TTC21B mRNA levels ascompared to the non-targeting controls.

C) Physical Perturbation

This example demonstrates disruption of the CTCF anchorsequence-mediated conjunction by physically preventing CTCF binding atthe anchor sequence.

HEK293T cells are serially transfected, first with plasmid encoding twodifferent dCas9 fusion proteins, then 8 hr later with one of the guideRNAs tiled around the anchor sequence (listed in Table 13) or a mixtureof both guide RNAs.

At 14 days post-transfection, cells are harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific; Thermo Fisher Scientific) and genomicDNA is extracted (Qiagen). The resulting cDNA is used for quantitativereal-time PCR (Thermo Fisher Scientific).

TTC21B-specific quantitative PCR probes/primers are multiplexed withinternal control quantitative PCR probes/primers as described in theprevious examples and gene expression is subsequently analyzed by a realtime PCR kit (Applied Biosystems, Thermo Fisher Scientific). Cellstransfected with guide RNAs proximal to the CTCF anchor sequence areexpected to show increases in TTC21B expression at 14 days aftermodification by either dCas9-DNMT3A-3L or dCas9-KRAB.

To determine differential CTCF binding at anchor sequences by targetedgRNAs and protein fusions versus non-targeting control gRNAs and proteinfusions, a CTCF chromatin immunoprecipitation-quantitative PCR assay(ChIP-qPCR) is performed. The CTCF ChIP protocol is performed asdescribed in previous examples. Phenol:chloroform purified DNA serves astemplate for SYBR Green (Thermo Scientific) qPCR using sequence-specificprimers (IDT) flanking the CTCF-binding sequence region. Diminishedinput-normalized amplification indicates reduced CTCF binding due to thetargeted physical disruptions.

Bulky effectors that physically disrupt CTCF binding at CTCF anchorsequences adjacent to the TTC21B gene demonstrate disruption of theanchor-mediated conjunction and increase TTC21B mRNA levels as comparedto the non-targeting controls.

Example 8: Disruption of a CTCF Anchor Sequence-Mediated Conjunction byGenetic Modification to Decrease Expression of the CDK6 Gene

The present Example demonstrates various strategies to decreaseexpression of a gene (in this case, CDK6) within a Type 1 anchorsequence-mediated conjunction. Among other things, the present Exampledemonstrates the successful modulation of gene expression by disruptionof the anchor sequence-mediated conjunction via, e.g., modification ofand/or perturbation at a CTCF anchor sequence.

Cyclin Dependent Kinase 6 (CDK6) is a member of the cyclin-dependentkinase (CDK) family. Cyclins are important regulators of cell cycleprogression. CDK6 is involved in regulation of cell proliferation bycontrolling a point of restriction in cell cycle. Dysregulation in CDK6has been found in 80-90% of tumors suggesting that modulation of CDK6activity might be relevant for cancer therapy. So far, development ofCDK6-specific small molecular inhibitors has been unsuccessful.

CDK6 is found within a CTCF anchor sequence-mediated conjunction. Thisconjunction also includes an associated transcriptional controlsequence, i.e. an enhancer, and is an example of Type 1 loop. Disruptionof the CTCF anchor sequence of the conjunction is expected to result indownregulation of CDK6.

Production of agents: All plasmids and guide RNAs (gRNA) have beenchemically synthesized from commercially available vendors. All agentswere reconstituted in sterile water. All sequences are provided in theMaterials and Methods section.

A) Genetic Perturbation

This example demonstrates disruption of the CDK6 gene Type 1 anchorsequence-mediated conjunction through genetic mutation of the putativeCTCF sites using CRISPR Cas9 technology.

TABLE 14 Sequences of gRNAs targeting putative CTCFanchor sequences associated with the CDK6 geneType 1 anchor sequence-mediated conjunction. IDGuide RNA Sequence (5′-3′) SACR-00046 CACATTAAAAATGTTACTAT SACR-00047TGTTTGAGTCAAACCTAAAA SACR-00048 ACGGTGGGTTCACGACTCAA SACR-00049AAAGTAACACTGCCATCTAA SACR-00050 AACACATAGAATCCATTAGA SACR-00051TGTGTTACTGCCATTGTCTG SACR-00052 TTAAATGTTGCCTCAGACAA SACR-00053AAAAACACAAAATAAGGTGG SACR-00054 AAATCAATCCAACAGATTAT

HEK293T cells were serially transfected with plasmid encoding Cas9 andeither a non-targeting gRNA (“Non-targeting,” where the guide RNAsequence has no homology to the human genome) or a gRNA, as listed inTable 14, targeted at or near the putative CTCF anchor sequencesassociated with the CDK6 gene. HEK293T cells were transfected first withplasmid encoding Cas9, and then 8 hr later with either a chemicallysynthesized gRNAs targeting the anchor sequence or a non-targeting gRNA(“Non-targeting,” where the guide RNA sequence has no homology to thehuman genome).

At 72 hr post-transfection, cells were harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific) and genomic DNA was extracted(Qiagen). The resulting cDNA was used for quantitative real-time PCR(Thermo Fisher Scientific).

CDK6-specific quantitative PCR probes/primers (Assay ID Hs01026371_m1,Thermo Fisher Scientific) were multiplexed with internal controlquantitative PCR probes/primers for PPIB (Assay ID Hs00168719_m1, ThermoFisher Scientific) using the FAM-MGB and VIC-MGB dyes, respectively, andgene expression was subsequently analyzed by a real time PCR kit(Applied Biosystems, Thermo Fisher Scientific). Cells transfected withguide RNA SACR-00046 showed about 30% decrease in CDK6 mRNA levels at 72hr relative to the “Non-targeting” control, (FIG. 13). Each biologicalreplicate is represented by empty box symbol.

Enzymatic effectors that modify DNA at or near the CTCF anchor sequenceassociated with the CDK6 gene demonstrated disruption of the CDK6 geneanchor-mediated conjunction and decrease CDK6 mRNA levels as compared tothe non-targeting controls

B) Epigenetic Modification

This example demonstrates disruption of the CDK6 gene associated CTCFanchor sequence-mediated conjunction by epigenetic modifications.

HEK293T cells are serially transfected, first with plasmid encodingeither dCas9-DNMT3A-3L (a fusion protein including the active domainsfrom a DNA methyltransferase) or dCas9-KRAB (a transcriptional repressorfusion protein), then 8 hr later with one of the gRNAs tiled around theanchor sequence (listed in Table 14) or a mixture of gRNAs tiled aroundthe anchor sequence.

At 72 hr post-transfection, cells are harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific) and genomic DNA is extracted(Qiagen). The resulting cDNA is used for quantitative real-time PCR(Thermo Fisher Scientific).

CDK6-specific quantitative PCR probes/primers are multiplexed withinternal control quantitative PCR probes/primers as described herein andgene expression is subsequently analyzed by a real time PCR kit (AppliedBiosystems, Thermo Fisher Scientific). Cells transfected with guide RNAsproximal to the CTCF anchor sequence are expected to show decreases inCDK6 expression at 72 hr after modification by either dCas9-DNMT3A-3L ordCas9-KRAB.

Effectors that target epigenetic modifications at or near the CTCFanchor sequence associated with the CDK6 gene demonstrate disruption ofthe CDK6 gene anchor-mediated conjunction to decrease CDK6 mRNA levelsas compared to the non-targeting controls.

C) Physical Perturbation

This example demonstrates disruption of CDK6 gene associated CTCF anchorsequence-mediated conjunction by physically preventing CTCF binding atthe anchor sequence.

HEK293T cells are serially transfected, first with plasmid encoding twodifferent dCas9 fusion proteins, then 8 hr later with one of the gRNAstiled around the anchor sequence (listed in Table 14) or a mixture ofgRNAs tiled around the anchor sequence.

At 72 hr post-transfection, cells are harvested for RNA extraction andcDNA synthesis using commercially available reagents and protocols(Qiagen; Thermo Fisher Scientific; Thermo Fisher Scientific) and genomicDNA is extracted (Qiagen). The resulting cDNA is used for quantitativereal-time

PCR (Thermo Fisher Scientific).

CDK6-specific quantitative PCR probes/primers are multiplexed withinternal control quantitative PCR probes/primers as described in theprevious examples and gene expression is subsequently analyzed by a realtime PCR kit (Applied Biosystems, Thermo Fisher Scientific). Cellstransfected with guide RNAs proximal to the CTCF anchor sequence areexpected to show decreases in CDK6 expression at 72 hr aftermodification by either dCas9-DNMT3A-3L or dCas9-KRAB.

To determine differential CTCF binding at anchor sequences by targetedgRNAs and protein fusions versus non-targeting control gRNAs and proteinfusions, a CTCF chromatin immunoprecipitation-quantitative PCR assay(ChIP-qPCR) is performed. The CTCF ChIP protocol is performed asdescribed in previous examples. Phenol:chloroform purified DNA serves astemplate for SYBR Green (Thermo Scientific) qPCR using sequence-specificprimers (IDT) flanking the CTCF-binding sequence region. Diminishedinput-normalized amplification indicates reduced CTCF binding due to thetargeted physical disruptions.

Bulky effectors that physically disrupt CTCF binding at CTCF anchorsequences associated with the CDK6 gene demonstrate disruption of theCDK6 gene anchor-mediated conjunction to decrease CDK6 mRNA levels ascompared to the non-targeting controls.

Example 9: Epigenetic Disruption of CTCF Binding in AnchorSequence-Mediated Conjunctions

The present Example demonstrates various therapeutic strategies thatincorporate disclosed methods and agents to epigenetically disrupt CTCFbinding in anchor sequence-mediated conjunctions.

A) Demethylation of a Specific CTCF Binding Motif for the TreatmentMuscular Dystrophy

Type 1 myotonic dystrophy (DM1), also known as Steinert disease, has asevere congenital form and a milder childhood-onset form as well as anadult-onset form. The gene implicated in DM1 is dmpk, whose gene productis a Ser/Thr protein kinase homologous to the MRCK p21-activated kinasesand the Rho family of kinases. The 3′ untranslated region of this genecontains 5-37 copies of a CTG trinucleotide repeat. Expansion of thisunstable motif to 50-5,000 copies causes myotonic dystrophy type I,which increases in severity with increasing repeat element copy number.Repeat expansion is associated with condensation of local chromatinstructure that disrupts the expression of genes in this region. Healthyhuman cells are enriched in CTCF bound to the CTCF sites flanking thedmpk repeat regions, whereas cells from DM1 patients lack CTCF binding(Cho et al., Antisense Transcription and Short Article Heterochromatinat the DM1 CTG Repeats Are Constrained by CTCF. Molecular Cell, Vol. 20,483-489 (2005).

In this example, a dCas9-TET1 fusion construct (using a Staphylococcusaureus dCas9) is designed with a sgRNA to target to the specific CTCFsites flanking the repeats at the DM1 locus. The construct is packagedin an adeno-associated virus (AAV) system, and is administeredsystemically (IV) to a subject having Steinert disease. A weeksubsequent to administration, site specific DNA methylation levels aremeasured in the subject: a sample of genomic DNA is taken from thesubject and analyzed by bisulphite analysis (Patterson et al., DNAMethylation: Bisulphite Modification and Analysis. J Vis Exp. 2011;(56): 3170). In addition, the sample is analyzed for transcription ofantisense and sense transcripts from the locus.

B) Restoration of Sodium Currents in a Cell Line Modeling SevereMyoclonic Epilepsy in Infancy by Disruption of CTCF Interactions toModulate a Type 2 Anchor Sequence-Mediated Conjunction

Voltage-gated Na+ channels in the brain are complexes of a 260-kDaα-subunit in association with auxiliary β-subunits (b1-b4) of 33 to 36kDa. The α-subunit includes the voltage sensors and the ion-conductingpore in four internally repeated domains (I-IV), each of which has sixα-helical transmembrane segments (S1-S6) and a pore loop that connectsS5 and S6. The association of β-subunits modifies the kinetics andvoltage dependence of gating, and these subunits are cell adhesionmolecules that interact with the extracellular matrix, other celladhesion molecules and the cytoskeleton. The type I sodium channel,NaV1.1, is the prototype of the voltage-gated sodium channel family inmammals. NaV1.1 is specifically localized in the neuronal cell body;NaV1.3 is abundant in the cell bodies of neurons during fetal andneonatal development but declines in adult rodents as the level ofNaV1.1 channels increases rapidly in the second postnatal week.

Voltage-gated sodium channels have crucial roles in the initiation andpropagation of action potentials and are crucial regulators of neuronalexcitability. Mutations in the NaV1.1 channel gene, SCN1A, causegenetically distinct epilepsy syndromes. Severe myoclonic epilepsy ininfancy (SMEI) is linked to de novo loss-of-function mutations in theSCN1A gene, which lead to haploinsufficiency of NaV1.1 channels. Thisrare convulsive disorder begins during the first year of life, withseizures often associated with fever, and progresses to prolonged,clustered or continuous seizures and to status epilepticus. After thesecond year of life, patients develop psychomotor delay, ataxia andcognitive impairment. They have an unfavorable long-term outcome becauseof the ineffectiveness of antiepileptic drug therapy.

The SCN1A gene is located on Chromosome 2 within a CTCF bound loop,whereas the upstream anchor is within 166,800,000-166,850, 000(GRCh37/hg19 assembly, see below), which separates it from an upstreamenhancing sequences. Disruption of the interaction between CTCF and itsanchor site on coordinates 166,800,000-166,850,000 enable the upstreamenhancing sequences to interact with SCN1A and upregulate itstranscription.

To disrupt the interaction between CTCF and its anchor site oncoordinates 166,800,000-166,850,000, the anchor site is methylated bytargeting dCas9-DNMT3a. The CTCF upstream of the SCN1a gene inchromosome 2, which in MCF7 cells and K562 is located within coordinates166810549-166810939, and the downstream CTCF site is located withincoordinates 166981175-166990179.

PCR amplified Dnmt3a from pcDNA3-hDNMT3A (Addgene plasmid: 35521) iscloned in modified pdCas9 plasmid (Addgene plasmid: 44246) with BamHIand EcoRI sites. dCas9-NLS-Dnmt3a is PCR amplified and cloned into FUWvector (Addgene plasmid: 14882) with Ascl and EcoRI to packagelentiviruses. The gRNA expression plasmids are cloned by insertingannealed oligos into modified pgRNA plasmid (Addgene plasmid: 44248)with AarI site. All constructs are sequenced before transfection.Lentiviruses expressing dCas9-Dnmt3a, and gRNAs are produced bytransfecting HEK293T cells with FUW constructs or pgRNA constructstogether with standard packaging vectors (pCMV-dR8.74 and pCMV-VSVG)followed by ultra-centrifugation-based concentration. Virus titer (T)are calculated based on the infection efficiency for 293T cells, whereT=(P*N)/(V), T=titer (TU/ul), p=% of infection positive cells accordingto the fluorescence marker, N=number of cells at the time oftransduction, V=total volume of virus used. SCN1a cell lines are usedfor this experiment.

Briefly, cells are cultured for viral infection. Cells are analyzed 3days post-infection in this study.

Sodium currents are measured by electrophysiological recordings.Whole-cell patch-clamp recordings are carried out at room temperatureusing an Axopatch 200B amplifier (Axon Instruments) with PCLAMP 6software (Axon Instruments) in voltage- or current-clamp configuration.For voltage-clamp experiments, cell capacitance (Cm) is calculated fromCm ¼ Q/V, where Q is the charge measured by integrating the capacitativecurrent evoked by a hyperpolarizing 10-mV voltage step (V) from aholding potential of −70 mV. For other recordings, capacitative currentsare minimized using the amplifier circuitry. 70% prediction and 90%series resistance compensation are routinely used. The remaining linearcapacity and leakage currents are eliminated by P/4 subtraction.

The intracellular solution contains 177 mM N-methyl-D-glucamine, 40 mMHEPES, 4 mM MgCl2, 10 mM EGTA, 1 mM NaCl, 25 mM phosphocreatine-Tris, 2mM ATP-Tris, 0.2 mM Na2GTP and 0.1 mM leupeptin, adjusted to pH 7.2 withH2504.

The extracellular solution for the recording of peak Na+ currentscontains 20 mM NaCl, 116 mM glucose, 10 mM HEPES, 1 mM BaCl2, 2 mMMgCl2, 55 mM CsCl2, 1 mM CdCl2, 1 mM CaCl2 and 20 mM tetraethylammoniumchloride, adjusted to pH 7.35 with NaOH.

Conductance-voltage (g-V) relationships (activation curves) arecalculated according to g ¼ INa/(V−ENa), where INa is the peak Na+current measured at potential V, and ENa is the calculated equilibriumpotential. Normalized activation and inactivation curves are fit toBoltzmann relationships of the form y ¼ 1/(1+exp[(V−V1/2)/k])+A, where yis normalized gNa or INa, A is the baseline conductance or current, V isthe membrane potential, V½ is the voltage of half-maximal activation(Va) or inactivation (Vh) and k is a slope factor. In fitting theactivation curves, A is fixed at 0. Analyses are carried out usingOrigin (Microcal) and pClamp (Axon Instruments).

For current-clamp experiments, cells are held at −80 mV, and theirfiring patterns are recorded in response to sustained depolarizations orhyperpolarizations (duration, 800 ms; increments, ±10 pA). Theinput-output relationship; action potential threshold, half-width, widthand peak, minimum voltage; and input resistance of cells are measured.The input-output relationship is defined as the dependence of the numberof action potentials generated upon the amplitude of current injection.The threshold is measured for the first action potential during thedepolarization protocol as the voltage corresponding to the peak of thethird differential of the action potential waveform. Action potentialhalf-width and width are measured at half-height and threshold,respectively. Input resistance is determined as the slope of the linearregression of the I-V plot for a series of hyperpolarizing pulses, whereI is current amplitude and V is steady-state voltage.

A successful intervention increases sodium current uponhyperpolarization.

Example 10: Physical Interference Between CTCF and its DNA AnchorSequence

The present Example demonstrates various therapeutic strategies thatincorporate disclosed methods and agents to disrupt CTCF binding inanchor sequence-mediated conjunctions.

A) Disruption of miR290 Anchor Sequence-Mediated Conjunction by PhysicalInterference

Polypeptide beta: PFDILYQ-GG-RGQGDC (SEQ ID NO: 3), and dCas9-TET1fusion as described in Xu, et al., Cell Discovery, 2015, 2):16009;doi:10.1038/celldisc.2016.9.

Experimental Design:

Peptides are synthesized using Fmoc solid-phase synthesis chemistry on aSymphony Peptide Synthesizer (Protein Technologies, Tucson, Ariz.). TheFmoc group (N-(9-fluorenyl)methoxycarbonyl) is removed by 20%piperidine, and Fmoc-amino acids are coupled using 0.1 M HBTU in DMFcontaining 0.4 M 4-methyl morpholine for 60 min. The resin-bound peptideis deprotected and cleaved from the resin using trifluoroacetic acid(TFA). Ethyl ether is added to precipitate the peptide from the TFAsolution. The precipitated peptide is then lyophilized.

The crude peptide is purified on a reversed-phase Vydac 218TP1010 C18column (Hesperia, Calif.) using a BioCad Sprint (Applied Biosystems,Foster City, Calif.). A flow rate of 10 mL/min with solvent A (0.1% TFAin deionized water) and solvent B (0.1% TFA in acetonitrile) is used.The column is equilibrated with 5% solvent B. After sample loading, thecolumn is eluted with a linear gradient from 5% solvent B to 100%solvent B in 60 min. The pure peptide fraction is identified bymatrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF MS). The mass peaks are observed that correlatewith the correct amino acid sequence.

The polypeptide beta is joined to dCas9-TET1 (Xu, et al., CellDiscovery, 2015, 2):16009; doi:10.1038/celldisc.2016.9) through clickchemistry.

To prepare for the click reaction, polypeptides are labeled with DBCO(Glen Research, Sterling, Va.). DBCO-sulfo-NHS ester is dissolved at aconcentration of 5.2 mg per 60 μL in water or anhydrous DMSO. This stocksolution is used to conjugate the amino-modified polypeptides in sodiumcarbonate/bicarbonate conjugation buffer, pH=˜9.

For a 0.2 μmol synthesis of polypeptide, polypeptide is dissolved in 500μL of conjugation buffer. Approx. a 6 fold excess (6 μL) ofDBCO-sulfo-NHS ester solution is added to the dissolved polypeptide. Themixture is vortexed and incubated at room temperature for 2-4 hours upto about overnight. The conjugated polypeptide is desalted on adesalting column (Glen Research, Sterling, Va.) to remove salts andorganics.

dCas9-TET1 fusion is resuspended in 500 μL of conjugation buffer.Approx. a 6 fold excess (6 μL) of azide solution is added to dCas9-TET1fusion. The mixture is vortexed and incubated at room temperature for2-4 hours up to about overnight. The conjugated fusion is desalted on adesalting column (Glen Research, Sterling, Va.) to remove salts andorganics.

For the click reaction, 1 mg of azide fusion is dissolved in 150 μL ofDMSO. The azide-fusion is added to 10 OD of DBCO conjugated polypeptidein 100 μL of water. The mixture is incubated at room temperatureovernight. The ligated fusion and polypeptides are desalted on adesalting column (Glen Research, Sterling, Va.) to remove salts andorganics.

This example demonstrates physical interference of gene expression withpolypeptides that target CpG dinucleotides of a gene.

Gene regulatory elements and their target genes generally occur withinanchor sequence-mediated conjunctions, chromosomal loop structuresformed by the interaction of two DNA sites bound by the CTCF protein andoccupied by the cohesin complex. Anchor sequence-mediated conjunctionsfor specific enhancing sequence-gene interactions are essential for bothnormal gene activation and repression, and form a chromosome scaffoldthat is largely preserved throughout development. Anchorsequence-mediated conjunctions are perturbed genetically andepigenetically in order to alter gene transcription in a targetedmanner. This is achieved by methylation (loop disruption) andde-methylation (promotes loop formation) of CpG dinucleotides on a CTCFbinding motif (CCGCGNGGNGGCAG, SEQ ID NO: 4), and by genome editing ofthe aforementioned sequence. Alternatively, a loop is disrupted byphysical interference with the CTCF-anchor sequence interaction.

Therapeutic Design:

This approach is tested experimentally by targeting the CTCF anchorsequences of the miR290 loop, a loop with activating polarity, thatharbors a super-enhancing sequence in mouse embryonic stem cells(mESCs). The polypeptide beta fusion with dCas9-TET1 includes sequencespecific polynucleotides that bind the two CTCF sites to physicallyinterfere (mediated by the polypeptide backbone and the polynucleotidesequence) with the looping function of CTCF, see FIG. 14.

Experimental Design:

In this experimental system, mouse embryonic stem cells are cultured onirradiated mouse embryonic fibroblasts (MEFs) with standard ESCs medium:(500 ml) DMEM supplemented with 10% FBS (Hyclone), 10 ug recombinantleukemia inhibitory factor (LIF), 0.1 mM b-mer-captoethanol(Sigma-Aldrich), penicillin/streptomycin, 1 mM L-glutamine, and 1%nonessential amino acids (all from Invitrogen), and exposed to thepolypeptides in their growth medium. After 2, 4, 6 h of exposure, mRNAis extracted from cells and analyzed for transcript number by RT-PCR:Cells are harvested using Trizol followed by Direct-zol (Zymo Research),according to manufacturer's instructions. RNA is converted to cDNA usingfirst-strand cDNA synthesis (Invitrogen SuperScript III). QuantitativePCR reactions are prepared with SYBR Green (Invitrogen), and performedin 7900HT Fast ABI instrument.

Successful interference causes an elevation of Nlrp12 gene, which isoutside of this super-enhancing sequence-containing anchorsequence-mediated conjunction and next to the targeted CTCF site,without affecting the expression of genes that are located inside themiR290 loop or of genes in other neighboring loops including A U018091and Myadm.

B) Nuclear Suppression of ELANE Transcription by Physical Interference

This example demonstrates ligating multiple polypeptide betas throughclick chemistry.

Click chemistry involves the rapid generation of compounds by joiningsmall units together via heteroatom links (C—X—C). The main objective ofclick chemistry is to develop a set of powerful, selective, and modularblocks that are useful for small- and large-scale applications. Theseclick reactions are bio-orthogonal, i.e. they can occur within organismswithout interfering with native biochemical processes. The reaction of adibenzylcyclooctyne (DBCO) linker with an azide linker to form a stabletriazole. This click reaction is very fast at room temperature, does notrequire a cytotoxic Cu(I) catalyst and creates stable triazoles. Thisunique covalent bond is created when DBCO, incorporated into one type ofbiomolecule, reacts with an azide linker, incorporated into a secondbiomolecule. The DBCO strain-promoted or Cu(I)-free [2+3] cycloadditionstrategy relies on the use of strained dibenzylcyclooctynes. Their usedecreases the activation energy for the cycloaddition click reaction,enabling it to be carried out without the need for catalysis at lowtemperatures with an efficiency greater than that of the Cu(I)-catalyzedligation.

Polypeptide beta is modified with dibenzylcyclooctyne (DBCO)modification and another polypeptide beta with an azide modification.

Experimental Design:

In the click reaction, succinimidyl esters, (5/6-carboxyfluoresceinsuccinimidyl ester andsuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate, Thermo FisherScientific, Waltham, USA) are dissolved in dry DMSO (Acros, Geel,Belgium). Primary amine labeling is carried out at 4° C. for 1 hour in20 mM Na Phosphate buffer pH 7.2 containing 0.05% dodecyl maltoside.

Maleimides, dibenzylcyclooctyne-PEG4-maleimide and azido-PEG3-maleimide(Jena Bioscience), are dissolved in dry DMSO. Sulfhydryl labeling isperformed at 25° C. for 2 hours in 20 mM Na Phosphate buffer pH 7.2containing 0.05% dodecyl maltoside. Copper-free coupling by clickchemistry is performed in the same buffer for 10 hours at 4° C.

After the reaction with 5/6-carboxyfluoresceine succinimidyl ester andthe maleimides, the labeled protein is separated from unreacted labelusing spin columns (Micro Biospin TM6 columns, Bio-Rad, Hercules, USA),according to the manufacturer's instructions.

Reaction products after coupling are analyzed by HPLC. 20-40 μl samplesare injected and separated on a chromatography system equipped with ananalytical column (300 mm×4.60 mm) eluted with 20 mM Na Phosphate bufferpH 7.2 containing 0.05% dodecyl maltoside at a flow rate of 0.5 ml/minand followed by absorption at 280 nm. Absorption spectra of peaks areobtained from the integrated spectral detector (Agilent technologiesG1315D diode array detector).

This example demonstrates inhibition of gene expression withpolypeptides that target an anchor sequence associated with the ELANEgene.

ELANE-related neutropenia includes severe congenital neutropenia (SCN)and cyclic neutropenia, both of which are primary hematologic disorderscharacterized by recurrent fever, skin and oropharyngeal inflammation(i.e., mouth ulcers, gingivitis, sinusitis, and pharyngitis), andcervical adenopathy. Infectious complications are generally more severein congenital neutropenia than in cyclic neutropenia and can lead todeath if untreated. Most cases of SCN respond to treatment withgranulocyte colony-stimulating factor, which increases the neutrophilcount and decreases the severity and frequency of infections. However,after 15 years with granulocyte colony stimulating factor treatment, therisk of developing myelodysplasia (MDS) or acute myelogenous leukemiaAML is approximately 15%-25%.

Mutations in the neutrophil elastase gene, ELANE, are the most commoncause of severe congenital neutropenia as well as of cyclic neutropenia.ELANE maps to 19p13.31 and mutations in the ELANE gene are identified inapproximately 35-84% of individuals with SCN. SCN and cyclic neutropeniasecondary to mutations in ELANE are inherited as autosomal dominantconditions. ELANE consists of five exons and encodes a 218 amino acidprotein known as neutrophil elastase (NE). NE belongs to the class ofserine proteases and is expressed exclusively in mature myelomonocyticcells and their committed immature precursors (promyelocytes andpromonocytes). Stored as an active protease in azurophilic granules, NEis released upon exposure of the neutrophil to inflammatory stimuli. Inthe extracellular environment, NE cleaves extracellular matrix proteins,while serine protease inhibitors antagonize the proteinase activity

Therapeutic Design:

In this example, the phenotype is reversed by silencing thetranscription of the ELANE gene in neutrophil precursors. In order toachieve that, the multimerized polypeptide betas are hybridized to anucleic acid sequence complimentary to an anchor sequence associatedwith the ELANE gene (e.g. caacggccgggccaaggctgtcgcaagaac, SEQ ID NO: 5),see FIG. 15, and delivered to myelomonocytes, promyelocytes andpromonocytes. The polypeptide-oligonucleotide passes through the cellmembrane and the nuclear membrane to hybridize to its target the anchorsequence, thereby disrupting the anchor sequence-mediated conjunctionthat harbors the ELANE gene, and the polypeptide-oligonucleotide hybridphysically interferes with the anchor sequence-mediated conjunction, andtherefore decreases the expression of ELANE.

Experimental Design:

This approach is tested in iPSC derived from SCN patients. To determineif gene correction of ELANE mutations restores granulopoieticdifferentiation, the SCN iPSCs are exposed to polypeptides containing anucleic acid sequence that complements the ELANE ORF, or a scrambledsequence, and selected for incorporation of the polypeptide. iPSCs aredifferentiated into CD45⁺CD34⁺ hematopoietic progenitors by 10 days ofculture in myeloid expansion medium (IMDM+Ham's F12 at 3:1 ratio)containing 0.5% N2 supplement, 1% B27 supplement without vitamin A, 0.5%human serum albumin, 100 μM monothioglycerol, 50 μg/ml ascorbic acid,100 ng/ml recombinant SCF, 10 ng/ml IL-3, and 10 ng/ml GM-CSF. Thecultures are further differentiated using granulopoietic cultureconditions (IMDM+Ham's F12 at 3:1 ratio) containing 0.5% N2 supplement,1% B27 supplement without vitamin A, 0.5% human serum albumin, 100 μMmonothioglycerol, 50 μg/ml ascorbic acid, and 50 ng/ml G-CSF (Neupogenfilgrastim) for 5 days. At the granulopoietic differentiation stage,cells are cultured at low (50 ng/ml) or high (1,000 ng/ml) G-CSF doses.During myeloid expansion and granulopoietic differentiation, cells arecultured in presence or absence of Sivelestat (Sigma-Aldrich) at aconcentration of 230 nM (˜5 times the IC50 for NE). At the end ofgranulopoietic differentiation, cells are cytospun onto a SuperfrostPlus Microscope slide (Fisher Scientific). The cells are Wright-Giemsastained and then scored for myeloid cell types (promyelocytes,myelocytes, metamyelocytes, bands, neutrophils, and monocytes) using anupright microscope (Motic BA310). For sorting the promyelocytes, cellsat the end of myeloid expansion are stained for CD45-Pacific Blue,CD34-PECy7, CD33-APC, CD11b-APCCy7 (catalog 557754, clone ICRF44, BDBiosciences), and CD15-FITC (catalog 562370, clone W6D3, BDBiosciences). The promyelocytes/myelocyte population (defined asCD45⁺/CD34⁻/CD33⁺/CD11b⁻/CD15^(dim)) is selected by FACS.

Expression of ELANE is quantitatively measured by PCR and determined tobe greater than untreated cells.

Example 11: Generation of Novel Anchor Sequence-Mediated Conjunctions

A) Generation of Novel Anchor Sequence-Mediated Conjunctions byHybridization of Methylated DNA with Exogenous UnmethylatedPolynucleotide-Polypeptide Effectors

This example demonstrates modulation of gene expression to createallele-specific anchor sequence-mediated conjunctions.

Gene regulatory elements and their target genes generally occur withinanchor sequence-mediated conjunctions, chromosomal loop structuresformed by the interaction of two DNA sites bound by the CTCF protein andoccupied by the cohesin complex. Anchor sequence-mediated conjunctionsprovide for specific enhancing sequence-gene interactions, are essentialfor both normal gene activation and repression, and form a chromosomescaffold that is largely preserved throughout development. Anchorsequence-mediated conjunctions are perturbed genetically andepigenetically in order to alter gene transcription in a targetedmanner. This is achieved by methylation (loop disruption) andde-methylation (promotes loop formation) of CpG dinucleotides on theCTCF binding motif (CCGCGNGGNGGCAG, SEQ ID NO: 4), and by genome editingof the aforementioned sequence. Alternatively, a loop is generated bythe targeted, exogenous delivery of a specific DNA strand and serves asan anchor sequence for CTCF.

The H19-IGF2 locus locus shows parent-of-origin specific loopconformations: An anchor sequence-mediated conjunction on the maternalallele allows an enhancing sequence-promoter interaction that activatesthe H19 gene, but not the IGF2 gene, which is excluded from the anchorsequence-mediated conjunction. A larger anchor sequence-mediatedconjunction is formed on the paternal allele to allow an enhancingsequence-promoter interaction that activates the IGF2 gene. Paternalallele-specific DNA methylation of a CTCF site in the H19 promoterregion abrogates CTCF binding, thus causing differential CTCF-CTCF loopformation that decreases H19 expression. Individuals who lose theseallele-specific anchor sequence-mediated conjunctions developBeckwith-Wiedemann syndrome (when both alleles have the paternal type ofanchor sequence-mediated conjunction) or Silver-Russell syndrome (whenboth alleles have the maternal type of anchor sequence-mediatedconjunction).

Therapeutic Design:

One polypeptide beta is designed to contain a double stranded,unmethylated CTCF anchor sequence with specificity to target the CTCFanchor sequences in the H19-IGF2 locus. See FIG. 16. The polypeptidedescribed herein mimics an unmethylated CTCF binding motif on one of thepaternal alleles to form a maternal type of loop in cells from patientswith Beckwith-Wiedemann syndrome caused by uniparental disomy.

Experimental Design:

In this experiment, skin fibroblasts derived from Beckwith-Widemannpatients are plated in standard primary fibroblast medium: (500 ml) DMEMsupplemented with 15% FBS (Hyclone), 0.1 mM b-mer-captoethanol(Sigma-Aldrich), penicillin/streptomycin, 1 mM L-glutamine, and 1%nonessential amino acids (all from Invitrogen), and exposed to thepolypeptides in their growth medium. After 2, 4, 6 h of exposure, mRNAis extracted from cells and analyzed for transcript number by RT-PCR:Cells are harvested using Trizol followed by Direct-zol (Zymo Research),according to manufacturer's instructions. RNA is converted to cDNA usingFirst-strand cDNA synthesis (Invitrogen SuperScript III). QuantitativePCR reactions are prepared with SYBR Green (Invitrogen), and performedin 7900HT Fast ABI instrument.

A successful manipulation causes an elevation of H19 gene expression,usually silent in the paternal allele.

B) Treatment of Fragile X Syndrome by Creating a Novel AnchorSequence-Mediated Conjunction

Fragile X is the leading cause of inherited intellectual disability. Itis caused by the amplification of a CGG repeat in the FMR1gene on the Xchromosome. The amplification causes DNA methylation of the CpGdinucleotides within the repeat as well as in the neighboring sequence,and subsequent decrease in expression of the gene. It is believed thattranscriptional silencing of the FMR1 gene is responsible for thepathology characteristic of the disease.

In this example, the FMR1 gene is activated by moving it into an anchorsequence-mediated conjunction that includes an enhancing sequence. Toidentify such anchor sequence-mediated conjunction, a ChIA-PET analysisis first carried out, where CTCF bound DNA elements are mapped on thegenome. This data is then overlayed with genome wide analysis ofenhancing sequences, as defined by Acetlyation of H3K27 and DNAseIhypersensitivity analysis (Kundaje et al 2015). The location of CTCFbinding motifs in the proximity of FMR1 is then analyzed to identify theones that need to be removed in order to bring the nearest and strongestenhancing sequences in close proximity to the FMR1 gene. Once the targetCTCF is identified, one of three approaches is applied:

Abolition of the CTCF2 Anchor Sequence by DNA Methylation:

A dCas9-DNMT3a fusion is designed, with a guide or antisense DNAoligonucleotide that targets the CTCF site to be methylated.Staphylococcus Aureus Cas9 will be used, and the construct will beintroduced by electroporation to cells derived from Fragile X patientsor to a Fragile X patient. Targeted methylation of the relevant CTCFanchor sequences by DNMT3a would lead to the looping of FMR1 togetherwith enhancing sequences and subsequent activation. 48 h afterelectroporation, chromatin, genomic DNA and total mRNA will be preparedfrom the electroporated cells. ChIA-PET analysis will be carried out todetermine if a loop was formed, encompassing the FMR gene and theenhancing sequences. Bisulphite analysis will be then used to determinemethylation levels at the target CTCF as well as within the FMR1 gene.Transcriptional activity of FMR1 will be assessed by RT-PCR from totalRNA derived from the cells.

Genome Editing and Deletion of a CTCF2 Anchor Sequence:

Alternatively, genome editing is used to mutate CTCF2 and in this waybring FMR1 to the activating anchor sequence-mediated conjunction. Inthis case, a Sa CRISPR-Cas9 targeting the relevant CTCF is designed, andincorporated into cells or to a Fragile X patient by electroporation. 48h after the manipulation, genomic DNA is extracted and sequenced todetermine whether the target CTCF was modified. FMR1 transcription isdetermined by RT-PCR analysis of total mRNA.

Use of a Dominant Negative Form of CTCF and Competitive Inhibition ofBinding:

To block the CTCF binding motif by means of a dominant negativeeffector, a protein is designed, with the ability to recognize and bindthe CTCF anchor sequence, but with a mutated dimerization domain. Withthis purpose, a Zinc Finger array can be designed with target CTCFspecificity, fused to a dominant negative CTCF protein lacking thedimerization domain, and having a Flag peptide. DNA encoding for thefusion protein will be introduced to cells or to a Fragile X patient byelectroporation. ChIP analysis is carried out 48 h after theelectroporation with a Flag antibody, to determine binding of thedominant negative effector to the target CTCF. Further analysis iscarried out as described above, to determine FMR1 transcription levels.

All three approaches may lead to the effective abolition of CTCF2 andsubsequent co-looping of the nearest enhancing sequences with the FMR1gene.

Example 12: Exemplary Anchor Sequences

Those of skill in the art reading the specification will understandanchor sequences can, in some embodiments, vary to some degree fromknown anchor sequences and/or those anchor sequences disclosed in thepresent specification. For example, although the present specificationdiscloses CTCF binding sequences as, in some embodiments, having orcomprising a portion having the sequence of SEQ ID NO: 1 or SEQ ID NO:2, in some embodiments, an anchor sequence to which CTCF binds is avariant of SEQ I D NO: 1 or SEQ ID NOL 2. For example, the below tableshows the probabilities of each of the four bases at a given position inSEQ ID NO: 1 for a CTCF binding domain.

TABLE 15 Probabilities of bases appearing in a CTCF binding domainPosition A C G T 5 0.061 0.876 0.023 0.039 6 0.009 0.989 0.000 0.002 70.815 0.014 0.071 0.100 8 0.044 0.578 0.366 0.012 9 0.117 0.475 0.0530.355 10 0.933 0.012 0.035 0.020 11 0.005 0.000 0.991 0.003 12 0.3660.003 0.621 0.010 13 0.059 0.013 0.553 0.374 14 0.013 0.000 0.978 0.00915 0.062 0.009 0.852 0.078 16 0.114 0.806 0.006 0.074 17 0.409 0.0140.558 0.019

1-243. (canceled)
 244. A method of decreasing expression of a genewithin a cell, the gene being within an anchor sequence-mediatedconjunction that comprises a first anchor sequence, a second anchorsequence, and an internal enhancing sequence, the method comprising astep of: contacting the first and/or second anchor sequence with asite-specific disrupting agent, wherein the site-specific disruptingagent is or comprises: a synthetic nucleic acid that binds specificallyto the first and/or second anchor sequence relative to non-target anchorsequences within the cell with sufficient affinity that it competes withbinding of an endogenous nucleating polypeptide within the cell. 245.The method of claim 244, wherein the first and/or the second anchorsequence is located within 500 kb of an external silencing or repressivesequence.
 246. The method of claim 244, wherein the gene is separatedfrom the internal enhancing sequence by at least 300 base pairs. 247.The method of claim 244, wherein the anchor sequence comprises a bindingmotif selected from the group consisting of: CTCF binding motif, USF1binding motif, YY1 binding motif, TAF3 binding motif, and ZNF143 bindingmotif.
 248. The method of claim 247, wherein the anchor sequencecomprises a CTCF binding motif.
 249. The method of claim 248, whereinthe CTCF binding motif has the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.250. The method of claim 248, wherein the endogenous nucleatingpolypeptide is or comprises CTCF.
 251. The method of claim 244, whereinthe synthetic nucleic acid comprises one or more phosphothiolatelinkages.
 252. The method of claim 244, wherein the gene is FOXJ3. 253.The method of claim 252, wherein the synthetic nucleic acid has asequence that is or comprises a sequence in Table
 8. 254. The method ofclaim 244, wherein the cell is a mammalian cell.
 255. The method ofclaim 244, wherein the step of contacting comprises delivering acomposition comprising the site-specific disrupting agent to a mammaliancell.
 256. The method of claim 255, wherein delivering the compositioncomprises transfecting the cell with the composition.